Formulations containing a somatostatin receptor agonist

ABSTRACT

The invention further relates to methods of treatment comprising administration of such pre-formulations, to pre-filled administration devices and kits containing the formulations, to the use of an alkylammonium EDTA salt to reduce the decomposition of the lipid components and/or any active agent contained within the pre-formulation.

FIELD OF THE INVENTION

The present invention relates to formulation precursors(pre-formulations) that upon exposure to water or aqueous media, such asbody fluids, spontaneously undergo a phase transition thereby forming acontrolled release matrix. In particular, the invention relates topre-formulations and compositions comprising a somatostatin receptoragonist, said pre-formulations and compositions having an improvedresistance to oxidation.

BACKGROUND TO THE INVENTION

Many bioactive agents including pharmaceuticals, nutrients, vitamins andso forth have a “functional window”. That is to say that there is arange of concentrations over which these agents can be observed toprovide some biological effect. Where the concentration in theappropriate part of the body (e.g. locally or as demonstrated by serumconcentration) falls below a certain level, no beneficial effect can beattributed to the agent. Similarly, there is generally an upperconcentration level above which no further benefit is derived byincreasing the concentration. In some cases increasing the concentrationabove a particular level results in undesirable or even dangerouseffects.

Some bioactive agents have a long biological half-life and/or a widefunctional window and thus may be administered occasionally, maintaininga functional biological concentration over a substantial period of time(e.g. 6 hours to several days). In other cases the rate of clearance ishigh and/or the functional window is narrow and thus to maintain abiological concentration within this window regular (or even continuous)doses of a small amount are required. This can be particularly difficultwhere non-oral routes of administration (e.g. parenteral administration)are desirable or necessary, since self-administration may be difficultand thus cause inconvenience and/or poor compliance. In such cases itwould be advantageous for a single administration to provide activeagent at a therapeutic level over the whole period during which activityis needed.

Some patients undergoing treatment will typically require a therapeuticdose to be maintained for a considerable period and/or ongoing treatmentfor many months or years. Thus a depot system allowing loading andcontrolled release of a larger dose over a longer period would offer aconsiderable advantage over conventional delivery systems.

Certain of the formulations of the present invention generate anon-lamellar liquid crystalline phase following administration. The useof non-lamellar phase structures (such as liquid crystalline phases) inthe delivery of bioactive agents is now relatively well established. Amost effective lipid depot system is described in WO2005/117830, and ahighly preferred lipid depot is described in that document. However,there remains scope for achieving depot formulations having improvedperformance in several respects.

Lipid controlled-release delivery systems have been developed withactive agents including GLP-1 (WO2006/131730), somatostatin analogues(WO2006/075124), LHRH analogues (WO2006/075125), as well as non-peptidessuch as buprenorphine (WO2014/016428). Lipid systems are also of valuein treatment in their own right and need not include active agents. Forexample, the FDA approved oral liquid Episil® alleviates the pain causedby oral mucositis and other inflammatory conditions of the mouth byforming a lipid barrier in the oral cavity, but does not require anyactive agent.

A particularly versatile combination of lipids is glycerol dioleate(GDO) and phosphatidyl choline (PC). However, sustained releasedformulations can be produced with a wide variety of other lipidcomponents including tocopherol (WO2006/075123), derivatives of sorbitol(WO2016/102683), triglycerides (WO2016/066655), and a variety ofphospholipid components including phosphatidyl ethanolamines(WO2013/083459 and WO2013/083460).

Both the lipid components, particularly unsaturated lipids, and anyactive agent contained in the pre-formulation or sustained releasecomposition are susceptible to oxidation, either during storage or invivo. It is desirable to decrease the extent of oxidation sinceoxidation processes may reduce the content of active agent and/orcontribute to the formation of unwanted decomposition products. This inturn reduces the shelf life of a product.

One particular factor contributing to oxidation in lipid compositions isthe presence of trace amounts of metal ions, particularly transitionmetals such as iron (Fe). Even when the lipid components are of highpurity grade it is often difficult to entirely remove traces of suchions. It is thought that equipment used for the manufacture of lipidformulations commonly includes stainless steel which can leach smallamounts of metal ions (particularly Fe) into the mixture. It istherefore common to include an antioxidant in lipid formulations. Thesegenerally function by chelating any metal ions, thereby hindering theirparticipation in oxidation processes.

It is a prerequisite that any antioxidant must be soluble in the lipidpre-formulation. It is described in WO2012/160213 that a carefullycontrolled amount of water can be included in lipid pre-formulationswithout causing a phase change into a liquid crystalline phase. Inpre-formulations containing an appreciable aqueous content, it may bepossible to include an effective amount of a water-soluble antioxidantsuch as ascorbic acid, inorganic salts of metal chelators, such asethylenediaminetetraacetic acid (EDTA) (e.g. sodium or calcium salts)and citric acid. However, for certain active agents it may be necessaryto avoid prolonged exposure to water during storage (e.g. because theactive agent is moisture sensitive), or a more desirable release profilemay be obtained without the inclusion of water in the pre-formulation.The present inventors have established that certain somatostatinreceptor agonists are less stable in formulations containing water. Theavoidance of water may also reduce the amount of trace metals which maybe present, since metal ions are generally more soluble in water than inan organic solvent or lipid environment. In lipid formulations having alow water content it is not possible to use conventional water-solubleantioxidants since these may not have the requisite solubility in asubstantially water-free lipid environment. It would therefore beadvantageous to provide an antioxidant which is soluble in asubstantially water-free lipid environment and which limits or preventsthe oxidative degradation of the lipid components of thepre-formulation, and any active agent contained within. This isparticularly the case for metal chelating agents such as EDTA where thestandard inorganic salts (sodium or calcium) are non-soluble or havenegligible solubility in non-aqueous environments (e.g. lipid matrices).

WO2010/020794 describes thiolated antioxidants as offering particularadvantages in lipid systems and suggests that these are also suitable innon-aqueous lipid systems. However, for certain end uses the presence ofa thiolated antioxidant may not be acceptable. This particularlyapplies, for example, to peptides or proteins having thiolated groups ordisulphide bridges. WO2010/020794 also mentions the possibility ofincluding EDTA or the sodium, disodium and calcium disodium salts ofEDTA as chelating agent although this is not an option which isexemplified. The present inventors have established that EDTA or thecommon salts thereof are not soluble to any appreciable extent in thetypes of lipid formulations described in WO2010/020794, i.e. those basedon GDO, SPC and an organic solvent such as ethanol.

It has now surprisingly been established that effective amounts ofalkylammonium salts of EDTA can be dissolved in a non-aqueous lipidenvironment, and that the resulting pre-formulations are highlyresistant to oxidative decomposition during storage. Furthermore,although alkylammonium EDTA salts are believed to have an effect ondecreasing the decomposition by the expected mechanism of sequesteringmetal ions, the present invention may in some embodiments improveoxidation resistance above the level that can be accounted for solely bythis mechanism.

The inventors have established that the inclusion of alkylammonium EDTAsalts can prevent, or substantially decrease the rate of, oxidation of awide variety of lipid components and/or active agents contained therein.The inventors have found that the inclusion of alkylammonium EDTA cansubstantially reduce the loss of assay of somatostatin receptor agonistsin drug samples tested in stability studies and thus increasesshelf-life of the drug product. EDTA salts have the advantage that theyare inexpensive, easily produced with a wide variety of countercations,and are generally regarded as safe (and are widely used e.g. inpharmaceutical applications).

The stabilizing and shelf-life extending effect of alkylammonium EDTA asfound by the inventors may be not only related to the prevention orreduction of oxidation reactions but may be also related to theprevention or reduction of other chemical degradation reactions, e.g.hydrolysis, acylation, deamidation.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical formulation comprisingan appropriate combination of lipid excipients, organic solvent,alkylammonium EDTA salt and a somatostatin receptor agonist that can beused as a depot-precursor formulation (referred to herein for brevity asa pre-formulation) to address one or more of the needs described above.

In a first aspect, the invention therefore provides a pre-formulationcomprising:

-   -   a) at least one di-acyl lipid; b) at least one phospholipid;    -   c) at least one biocompatible, organic solvent;    -   d) an alkyl ammonium EDTA salt; and    -   e) at least one somatostatin receptor agonist;        wherein the pre-formulation has a water content in the range of        0 to 1.0 wt %.

Pre-formulations according to the invention preferably form, or arecapable of forming, at least one liquid crystalline phase structure uponcontact with excess aqueous fluid.

In a second aspect, the invention provides a pre-formulation comprising:a lipid controlled-release matrix comprising:

-   -   a) at least one of a di-acyl lipid;    -   b) at least one phospholipid;    -   c) at least one biocompatible, organic solvent;    -   d) an alkyl ammonium EDTA salt;    -   e) at least one somatostatin receptor agonist;        wherein the pre-formulation has a water content in the range of        0 to 1.0 wt %;        wherein the pre-formulation forms, or is capable of forming, at        least one liquid crystalline phase structure upon contact with        excess aqueous fluid.

A particularly preferred combination of components is glycerol dioleate(GDO), phosphatidyl choline (PC), ethanol, tetrakis(ethanolammonium)EDTA, and octreotide.

The pre-formulations are highly useful for the controlled and sustainedrelease of an active agent, especially those requiring or benefitingfrom a very flat release profile and/or minimal “burst” uponadministration. In a corresponding embodiment, the invention thereforeprovides for a mixture of:

a lipid controlled-release matrix comprising:

-   -   a) at least one of a di-acyl lipid;    -   b) at least one phospholipid;    -   c) at least one biocompatible, organic solvent;    -   d) an alkyl ammonium EDTA salt; and    -   e) at least one somatostatin receptor agonist;        wherein the pre-formulation has a water content in the range of        0 to 1.0 wt %;

in the manufacture of a pre-formulation for use in the sustainedadministration of said active agent. In a preferred embodiment, thepre-formulation forms, or is capable of forming, at least one liquidcrystalline phase structure upon contact with excess aqueous fluid.

A “a somatostatin receptor agonist” as referred to herein, may be anycompound having agonistic function at one or more somatostatin receptors(SSTRs). There are five known types of SSTRs (SSTR1-SSTR5), showingequally high affinity for SST-14 (see below for description of SST-14).The most investigated somatostatin receptor agonists, includingoctreotide, show high selectivity for SSTR2 and SSTR5. Thus in onepreferred embodiment, somatostatin agonists as indicated herein have anagonistic function at somatostatin receptors including SSTR2 and/orSSTR5.

Preferred somatostatin receptor agonists herein are SST-14, SST-28,octreotide, lanreotide, vapreotide, pasireotide (SOM230) and relatedpeptides. More preferred somatostatin receptor agonists herein areoctreotide and pasireotide (SOM230). Most preferred somatostatinreceptor agonist herein is octreotide.

Typically the somatostatin receptor agonist will be formulated at alevel sufficient to provide an in vivo concentration at a functionallevel. The somatostatin receptor agonist may be either a natural orsynthetic somatostatin receptor agonist which provides a therapeutic,palliative and/or prophylactic effect when administered to a suitablesubject (typically being one in need of such an effect).

In a further embodiment, the invention therefore provides a method forthe treatment of a human or non-human mammalian subject comprisingadministering to said subject a pre-formulation as described herein.Such a method may be for the treatment of a human or non-human mammaliansubject in need thereof to combat, (e.g. cure, improve, prevent orameliorate the symptoms of) at least one condition selected fromacromegaly, cancers, carcinomas, melanomas, tumours expressing at leastone somatostatin receptor, sst(2)-positive tumours, sst(5)-positivetumours, prostate cancers, gastro-entero-pancreatic endocrine tumours,gastro-entero-pancreatic neuroendocrine (GEP NE) tumours (GEP-NET), lungneuroendocrine tumours (lung NET), carcinoid tumours, insulinomas,gastrinomas, vasoactive intestinal peptide (VIP) tumours andglucagonomas, TSH-secreting pituitary adenomas, elevated growth hormone(GH), elevated insulin-like growth factor I (IGF-I), varicial bleeding(especially espohageal), chemotherapy induced gastro intestinal problems(such as diarrhea), lymphorrhea, diabetic retinopathy, thyroid eyedisease, obesity, pancreatitis, and related conditions.

The pre-formulations as described herein for use in such methods form afurther aspect of the invention.

Correspondingly, in a further aspect, the present invention provides theuse of a low viscosity pre-formulation comprising a mixture of:

-   -   a) at least one di-acyl lipid;    -   b) at least one phospholipid;    -   c) at least one biocompatible, organic solvent;    -   d) an alkylammonium EDTA salt; and    -   e) at least one somatostatin receptor agonist;        wherein the pre-formulation has a water content in the range of        0 to 1.0 wt %;        in the manufacture of a low viscosity pre-formulation medicament        for use in the in vivo formation of a depot for treatment of at        least one condition selected from acromegaly, cancers,        carcinomas, melanomas, tumours expressing at least one        somatostatin receptor, sst(2)-positive tumours, sst(5)-positive        tumours, prostate cancers, gastro-entero-pancreatic endocrine        tumours, gastro-entero-pancreatic neuroendocrine (GEP NE)        tumours, lung NE tumours (lung NET), carcinoid tumours,        insulinomas, gastrinomas, vasoactive intestinal peptide (VIP)        tumours and glucagonomas, TSH-secreting pituitary adenomas,        elevated growth hormone (GH), elevated insulin-like growth        factor I (IGF-I), varicial bleeding (especially espohageal),        chemotherapy induced gastro intestinal problems (such as        diarrhea), lymphorrhea, diabetic retinopathy, thyroid eye        disease, obesity, pancreatitis, and related conditions.

One of the advantages of the formulations of the present invention overmany other controlled-release compositions is that they are stable tostorage in their final form and thus little or no preparation isrequired at the time of administration. This allows the pre-formulationsto be ready-to-administer and also to be supplied in convenient,ready-to-administer form. In a further aspect, the invention thereforeprovides a pre-filled administration device containing a pre-formulationas described herein. Such a device will generally provide either asingle administration or multiple administrations of a composition whichwill deliver, for example, a dosage of active agent in the range of 1 μgto 15 mg/day, such as 0.1 mg to 15 mg/day or 1 μg to 5 mg/day.

In a further aspect the invention provides a kit comprising saidadministration device according to the invention.

The kit can optionally contain instructions for subcutaneous orintramuscular administration of said pre-formulation. Allpre-formulation described herein are suitable for use in such a kit andmay thus be contained therein.

The kits of the invention can optionally include additionaladministration components such as needles, swabs, and the like and willoptionally contain instructions for administration.

BRIEF SUMMARY OF THE ATTACHED FIGURES

FIG. 1. Octreotide assay of Samples 53-54 as a function of time atstorage conditions 25° C./60% RH and 40° C./75% RH.

FIG. 2. Octreotide assay of Samples 55-60 as a function of EDTAconcentration (0-750 ppm or 0-0.075 wt %) at three time points (0, 1 and2 months) after storage at 40° C./75% RH.

FIG. 3. OCT assay in SPC/GDO/EtOH/PG based formulations in the absence(Sample 61) and presence of 100 ppm EDTA (Sample 62) as a function oftime at 25° C./60% RH. Formulations were stored in pre-filled glasssyringes.

FIG. 4. OCT assay in SPC/GDO/EtOH/PG based formulations as a function ofFe³⁺ concentration in the presence of 0, 25, 100 and 250 ppm EDTA(Samples 63-78) recorded at 1 month of storage at 40° C./75% RH.Formulations were stored in vials with ambient air in the headspace.

FIG. 5. OCT assay data in SPC/GDO/EtOH/PG formulations as a functionEDTA:Fe³⁺ molar ratio after 1 month of storage at 40° C./75% RH.Formulations were stored in vials with ambient air in the headspace.

FIG. 6. Assay (a) and Stability Index (b) values of OCT inSPC/GDO/EtOH/PG formulations as a function of time at 40° C./75% RH:without additives (Sample 79, reference), with EDTA(Na) (Sample 80),with EDTA(Na)/ETA (Sample 81), with EDTA (Sample 82), and with EDTA/ETA(Sample 83). Formulations were stored in vials with normal air in theheadspace. Except for the reference Sample 79, all formulations alsocontained 5 ppm Fe³⁺.

FIG. 7. OCT assay in SPC/GDO/EtOH/PG based formulations in the absence(Sample 79) and presence of 100 ppm EDTA solubilized in the lipidformulation by the use of ETA (Sample 84), DiETA (Sample 85) orethylenediamine (Sample 86) as a function of time at 40° C./75% RH.Formulations were stored in vials with normal air in the headspace.

FIG. 8. OCT assay in SPC/GDO/EtOH/PG (Sample 79—reference without EDTAand Sample 84 with 100 ppm EDTA) based formulations as a function oftime at 40° C./75% RH. Formulations were stored in vials with normal airin the headspace.

FIG. 9. SOM assay in SPC/GDO/EtOH/PG (Sample 89—reference without EDTAand Sample 90 with 100 ppm EDTA) based formulations as a function oftime at 40° C./75% RH (a) and 25° C./60% RH (b). Formulations werestored in vials with normal air in the headspace.

FIG. 10. Vial headspace oxygen concentration for SPC/GDO (50/50 w/w)based formulations without (Samples 103 and 104) and with 100 ppm EDTA(105 and 106) in the absence (a) and presence of 5 ppm Fe³⁺ (b) as afunction of time at 60° C./ambient RH. Formulations were stored in vialswith normal air in the headspace.

FIG. 11. Vial headspace oxygen concentration for SPC/GDO (35/65 w/w)based formulations without (Samples 107 and 108) and with 100 ppm EDTA(109 and 110) in the absence (a) and presence of 5 ppm Fe³⁺ (b) as afunction of time at 60° C./ambient RH. Formulations were stored in vialswith normal air in the headspace.

FIG. 12. Vial headspace oxygen concentration for SPC/GDO (50/50 w/w)based formulations without (Samples 103 and 104) and with 100 ppm EDTA(105 and 106) in the absence (a) and presence of 5 ppm Fe³⁺ (b) as afunction of time at 40° C./75% RH. Formulations were stored in vialswith normal air in the headspace.

FIG. 13. Vial headspace oxygen concentration for SPC/GDO (35/65 w/w)based formulations without (Samples 107 and 108) and with 100 ppm EDTA(109 and 110) in the absence (a) and presence of 5 ppm Fe³⁺ (b) as afunction of time at 40° C./75% RH. Formulations were stored in vialswith normal air in the headspace.

DETAILED DESCRIPTION OF THE INVENTION

The formulations of the present invention are lipid-based, aresubstantially non-aqueous and form a depot composition upon contact withan aqueous fluid. As used herein, the terms “formulation” or“pre-formulation” relate to the mixture of components (a), (b), (c), (d)and (e) which is typically of low viscosity. The term “depot” relates tothe composition which is formed upon exposure of the pre-formulation toexcess aqueous fluid, e.g. as occurs during numerous parenteraladministration routes. Without wishing to be bound by theory, it isthought that this change is brought about at least in part by exchangeof solvent (c) for aqueous fluid. The depot typically has a much higherviscosity than the corresponding formulation and provides for thegradual release of any active agent contained within the depot.

In a preferred aspect, the formulations of the present inventiongenerate a non-lamellar phase (e.g. non-lamellar liquid crystallinephase) following administration. The use of non-lamellar phasestructures (such as liquid crystalline phases) in the delivery ofbioactive agents is now relatively well established. A most effectivelipid depot system is described in WO2005/117830, and a suitable lipidmatrix for use in the present invention is described in that document,the full disclosure of which is hereby incorporated herein by reference.For a description of the most favourable phase structures of suchformulations, attention is drawn to the discussion in WO2005/117830 andparticularly to page 29 thereof. Preferably the pre-formulationaccording to the invention has an L₂ phase (liquid phase) structure oris a liquid solution or molecular solution.

All % are specified by weight herein throughout, unless otherwiseindicated. Percent (%) by weight may be abbreviated e.g. as wt %.Furthermore, the % by weight indicated is the % of the totalpre-formulation including all of the components indicated herein, unlessotherwise indicated. Where a percentage by weight is given in relationto component (e) the weight relates to the amount of free base (e.g.where a salt is used), unless otherwise indicated. In certain Examples,the wt % of a specified salt is provided but is indicated whereappropriate and may be readily converted to the corresponding weight offree base.

The pre-formulations can optionally consist of essentially only thecomponents indicated herein (including where appropriate additionaloptional components indicated herein below and in the attached claims)and in one aspect consist entirely of such components.

The lipid-based systems described herein comprise lipid components (a)and (b), an organic solvent (c), an alkylammonium EDTA salt (d), and asomatostatin receptor agonist (e).

The present inventors have now surprisingly established that byappropriate choice of antioxidant, the oxidation resistance of the lipidand any somatostatin receptor agonist(s) contained in thepre-formulation can be significantly improved.

Whilst various alkylammonium EDTA salts are known, for instance fromScott and Kyffin (Biochem. J. (1978) 169, 697-701), their use as anantioxidant in lipid systems and compatibility with such formulationshas been hitherto unknown. Scott and Kyffin describe the use of solubleEDTA salts in the demineralisation of bone samples, where EDTA acts as asequestering agent. A particularly suitable solution is said to be 80%aqueous ethanol containing 0.2 M trimethylammonium EDTA. No use in lipidformulations nor solubility in lipids is suggested. The purpose of theEDTA salt in the present invention is as a preservative or stabilityenhancing agent in lipid formulations, and is very different from thatdescribed previously.

Component a)—Neutral Lipid

Preferable ranges for component a) are 20-90 wt. %, preferably 30-70 wt.%, more preferably 33-60%, particularly 38 to 43%.

Component “a” as indicated herein is at least one di-acyl lipid (e.g. atleast one neutral di-acyl lipid (having no net charge at physiologicalpH)) comprising a polar “head” group and two non-polar “tail” groups.

As used herein, the term “acyl lipid” relates to a lipid componentcontaining a polyol “head” group and one or more apolar “tail groups”.In certain embodiments the polyol may be glycerol, a sugar or a hexitansuch as sorbitan. The term “hexitan” denotes a hexitol of formulaHOCH₂(CHOH)₄CH₂OH which has cyclised by formally losing one equivalentof water, to form a five or six membered ring, preferably a fivemembered furanose ring. Sorbitan is a particularly suitable “headgroup”, particularly as a component of a mono-acyl lipid component

In the case of di-acyl lipids, it is most preferred that the lipidcomponent comprises a glycerol head group with two apolar tail groups.The two non-polar groups may have the same or a differing number ofcarbon atoms and may each independently be saturated or unsaturated.Examples of non-polar groups include C₆-C₃₂ alkyl and alkenyl groups,which are typically present as the esters of long chain carboxylicacids. These are often described by reference to the number of carbonatoms and the number of unsaturations in the carbon chain. Thus, CX:Zindicates a hydrocarbon chain having X carbon atoms and Z unsaturations.Examples particularly include lauroyl (C12:0), myristoyl (C14:0),palmitoyl (C16:0), phytanoyl (C16:0), palmitoleoyl (C16:1), stearoyl(C18:0), oleoyl (C18:1), elaidoyl (C18:1), linoleoyl (C18:2), linolenoyl(C18:3), arachidonoyl (C20:4), behenoyl (C22:0) and lignoceroyl (C24:9)groups. Thus, typical non-polar chains are based on the fatty acids ofnatural ester lipids, including caproic, caprylic, capric, lauric,myristic, palmitic, phytanic, palmitolic, stearic, oleic, elaidic,linoleic, linolenic, arachidonic, behenic or lignoceric acids, or thecorresponding alcohols. Preferable non-polar chains are palmitic,stearic, oleic and linoleic acids, particularly oleic acid.

Mixtures of any number of diacyl lipids may be used as component a).Preferably this component will include at least a portion of C18 lipids(e.g. a diacyl glycerol (DAG) having one or more C18:0, C18:1, C18:2 orC18:3 non-polar groups), such as glycerol dioleate (GDO) and/or glyceroldilinoleate (GDL). A highly preferred example is DAG comprising at least50%, preferably at least 80% and even comprising substantially 100% GDO.

Since GDO and other diacyl glycerols may be derived from naturalsources, there is generally a certain proportion of “contaminant” lipidhaving other chain lengths etc. In this context, “pure” GDO is adi-ester of glycerol and two C18:1 fatty acids. Any other diacylglycerol is considered to be an impurity. In one aspect, GDO as usedherein is thus used to indicate any commercial grade of GDO withconcomitant impurities (i.e. GDO of commercial purity). These impuritiesmay be separated and removed by purification but providing the grade isconsistent this is rarely necessary. If necessary, however, “GDO” may beessentially chemically pure GDO, such as at least 70% pure, preferablyat least 75% pure and more preferably at least 80% pure GDO.Correspondingly, the C18:1 content of GDO referred to herein may bearound 80%, preferably at least 85% and more preferably at least 90%.

It will be appreciated that any material used, including component a),may potentially include unavoidable trace impurities of metals,optionally including heavy metals. According to the certificates ofanalysis for commercially available GDO (e.g. from Croda), a typicalmaximum concentration of heavy metals (or elemental impurities) in GDOis 5 ppm. Without being bound by theory, the common presence of thesemetal components and their sequestration in the various aspects of thepresent invention may be at least partially responsible for theadditional stability observed. However, a more common issue may be thepresence of iron ions, which may be absorbed from iron-based alloymaterials used in handling/storage of the materials.

Component b)—Phospholipid

Component b) in the preferred lipid matrices of the present invention isat least one phospholipid. Preferable ranges of component b) are 20-80wt. %, preferably 30-70 wt. %, more preferably 33-55% (e.g. 35-55%),particularly 38 to 43%. Ratios of a:b are typically 40:60 to 70:30,preferably 45:55 to 55:45 and more preferably 40:60 to 54:46, such as45:55 to 54:46 or 47:53 to 53:47. Ratios of around 50:50 (e.g. 49:51 to51:49) are highly effective in certain embodiments.

Preferred phospholipid polar “head” groups include phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol.Most preferred are phosphatidyl choline (PC) and phosphatidylethanolamine (PE), especially PC. As with component a), this componentcomprises a polar head group and non-polar tail group(s). The differencebetween components a) and b) lies principally in the polar group. Thenon-polar portions may thus suitably be derived from the fatty acids orcorresponding alcohols considered above for component a). Thephospholipid will contain two non-polar groups. Again, C18 groups arepreferred and may be combined with any other suitable non-polar group,particularly C16 groups.

The phospholipid portion may be derived from a natural source. In thecase of PC, suitable sources of phospholipids include egg, heart (e.g.bovine), brain, liver (e.g. bovine) and plant sources including soybean.Such sources may provide one or more constituents of component b, whichmay comprise any mixture of phospholipids. Any single PC or mixture ofPCs from these or other sources may be used, but mixtures comprising soyPC or egg PC are highly suitable. The PC component preferably containsat least 50% soy PC or egg PC, more preferably at least 75% soy PC oregg PC and most preferably essentially pure soy PC or egg PC.

In one embodiment applicable to all aspects of the invention, componentb) comprises PC. Preferably the PC is derived from soy. Preferably thePC comprises 18:2 fatty acids as the primary fatty acid component with16:0 and/or 18:1 as the secondary fatty acid components. These arepreferably present in the PC at a ratio of between 1.5:1 and 6:1. PChaving approximately 60-65% 18:2, 10 to 20% 16:0, 5-15% 18:1, with thebalance predominantly other 16 carbon and 18 carbon fatty acids ispreferred and is typical of soy PC.

In an alternative but equally preferred embodiment, the PC component maycomprise synthetic dioleoyl PC (DOPC). The use of DOPC may provideincreased stability and so will be particularly preferable forcompositions needing to be stable to long term storage, and/or having along release period in vivo. In this embodiment the PC componentpreferably contains at least 50% synthetic dioleoyl PC, more preferablyat least 75% synthetic dioleoyl PC and most preferably essentially puresynthetic dioleoyl PC. Any remaining PC is preferably soy or egg PC asabove.

Since the pre-formulations of the invention are to be administered to asubject it is important that the components are biocompatible. In thisregard, the preferred lipid matrices for use in the pre-formulations ofthe present invention are highly advantageous since PC and diacylglycerols are well tolerated and are broken down in vivo into componentsthat are naturally present in the mammalian body.

It will be appreciated that component b) may include unavoidable traceimpurities of heavy metals. According to the certificates of analysisfor commercially available soy PC (e.g. from Lipoid), a typical maximumconcentration of heavy metals (or elemental impurities) in soy PC is 10ppm.

Synthetic or highly purified PCs, such as dioleoyl phosphatidyl choline(DOPC) are highly appropriate as all or part of component b). Thesynthetic dioleoyl PC is most preferably1,2-dioleoyl-sn-glycero-3-phosphocholine, and other synthetic PCcomponents include DDPC (1,2-Didecanoyl-sn-glycero-3-phosphocholine);DEPC (1,2-Dierucoyl-sn-glycero-3-phosphocholine); DLOPC(1,2-Dilinoleoyl-sn-glycero-3-phosphocholine); DLPC(1,2-Dilauroyl-sn-glycero-3-phosphocholine); DMPC(1,2-Dimyristoyl-sn-glycero-3-phosphocholine); DOPC(1,2-Dioleoyl-sn-glycero-3-phosphocholine); DPPC(1,2-Dipalmitoyl-sn-glycero-3-phosphocholine); DSPC(1,2-Distearoyl-sn-glycero-3-phosphocholine); MPPC(1-Myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine); MSPC(1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine); PMPC(1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine); POPC(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine); PSPC(1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine); SMPC(1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine); SOPC(1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine); and SPPC(1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine), or any combinationthereof.

A particularly favoured combination of components a) and b) are GDO withPC, especially GDO with soy PC and/or DOPC. Appropriate amounts of eachcomponent suitable for the combination are those amounts indicatedherein for the individual components in any combination. This appliesalso to any combinations of components indicated herein, where contextallows.

Component c)—Biocompatible Organic Solvent

Component c) of the pre-formulations of the invention is at least onebiocompatible organic solvent. Since the pre-formulation is to generatea depot composition following administration (e.g. in vivo), typicallyupon contact with excess aqueous fluid, it is desirable that thissolvent be tolerable to the subject and be capable of mixing with theaqueous fluid, and/or diffusing or dissolving out of the pre-formulationinto the aqueous fluid. Solvents having at least moderate watersolubility are thus preferred. As will be described hereinafter,component c) may include a polar co-solvent.

Component c) comprises or consists of at least one solvent selected fromthe group consisting of: alcohols, amines, amides or esters. Preferablycomponent c) comprises at least a mono-alcoholic solvent. Mostpreferably component c) comprises ethanol, propanol, ispropanol, ormixtures thereof. It is particularly preferred the component c)comprises or consists of ethanol. Component c) may comprise or consistof a mono-alcoholic solvent, preferably ethanol, and a polar co-solvent.Mixtures comprising or consisting of ethanol and propylene glycol arealso highly preferred.

The amount of component c) in the pre-formulation will have aconsiderable effect upon several features. In particular, the viscosityand the rate (and duration) of release may alter significantly with thesolvent level. The amount of solvent will thus be at least sufficient toprovide a low viscosity mixture but will additionally be determined soas to provide the desired release rate. Typically a level of 1 to 30%,particularly 2 to 20% solvent will provide suitable release andviscosity properties. In some embodiments, levels of 2 to 18%, such as 2to 16%, especially 2 to 15% are preferred. In particularly preferredembodiments the amount of c) is 5 to 18%, such as 8 to 18%, such as 9 to17%, especially 11 to 15%.

As indicated above, the amount of component c) in the pre-formulationsof the invention will be at least sufficient to provide a low viscositymixture (e.g. a molecular solution) of components a) to e), and will beeasily determined for any particular combination of components bystandard methods.

The phase behaviour may be analysed by techniques such as visualobservation in combination with polarized light microscopy, X-rayscattering and diffraction techniques, nuclear magnetic resonance, andcryo-transmission electron microscopy (cryo-TEM) to look for solutions,L₂ or L₃ phases, or liquid crystalline phases or as in the case ofcryoTEM, dispersed fragments of such phases. Viscosity may be measureddirectly by standard means. As described above, an appropriate practicalviscosity is that which can effectively be syringed and particularlysterile filtered. This will be assessed easily as indicated herein.

A highly preferred combination for components a), b) and c) is GDO, soyPC and ethanol, especially GDO, soy PC and mixtures of ethanol andpropylene glycol. As indicated above, appropriate amounts of eachcomponent suitable for the combination are those amounts indicatedherein for the individual components, in any combination.

It is preferable that little or none of component c) contains halogensubstituted hydrocarbons since these tend to have lowerbiocompatibility.

Component c) as used herein may be a single solvent or a mixture ofsuitable solvents but will generally be of low viscosity. The viscosityof the “low viscosity” solvent component c) (single solvent or mixture)should typically be no more than 18 mPas at 20° C. This is preferably nomore than 15 mPas, more preferably no more than 10 mPas and mostpreferably no more than 7 mPas at 20° C.

It is described in WO2012/160213 that the addition of a polar solvent inaddition to a mono-alcoholic solvent results in numerous advantagesincluding reduced viscosity and reduced active agent burst profile. Inaddition to the preferred aspects described previously for component c),in one particularly preferred embodiment component c) comprises amono-alcoholic solvent and a polar co-solvent. The term “polarco-solvent” as used herein defines a solvent having a dielectricconstant (diel) of at least 28 at 25° C., more preferably at least 30 at25° C. but is not water or any aqueous fluid. Highly suitable examplesinclude propylene glycol (diel ˜32), and N-methyl-2-pyrrolidone (NMP,diel ˜32). The preferred levels of component c) recited herein applyequally to mixtures of mono-alcoholic solvent and a polar co-solventunless context permits otherwise.

In a particularly preferred embodiment component c) comprises, consistsessentially of, or consists of a mixture of a mono-alcoholic solvent anda polar co-solvent. The polar co-solvent may in one embodiment be adi-alcoholic C3-C6 organic solvent, i.e. a C3-C6 organic solventcomprising two hydroxy groups. The di-alcoholic solvent is preferablypropylene glycol. When present, a polar co-solvent is included at alevel of 2 to 12 wt. % of the pre-formulation, such as 3 to 10 wt. %,especially 4 to 9 wt. %. This level is counted as part of the rangesrecited above for component c). Most preferably component c) comprises,consists essentially of, or consists of a mixture of ethanol andpropylene glycol (PG).

Where both an organic mono-alcoholic solvent and a polar co-solvent arepresent, e.g. ethanol and PG, the ratio of mono-alcoholic solvent topolar co-solvent solvent is preferably in the range 20:80 to 70:30,preferably 30:70 to 70:30 (w/w), more preferably 40:60 to 60:40.Approximately equal amounts of mono- and di-alcoholic components arehighly appropriate.

In an especially preferred embodiment component c) is present at a levelof 1 to 30% and comprises, consists or consists essentially of a mixtureof ethanol and PG, wherein the ratio of ethanol to PG (w/w) is in therange of 30:70 to 70:30, preferably 40:60 to 60:40. More preferablycomponent c) is present at a range of 5 to 15 wt % or 8 to 18 wt %, mostpreferably 8-18% wt %, and is a mixture of ethanol and PG in a ratio of40:60 to 60:40 (w/w).

For the avoidance of doubt, even where a polar co-solvent is present inthe pre-formulations of the present invention, the total water levelwill remain as described in the various embodiments herein (e.g. 0.1 to1.0 wt %).

Component d)—Alkylammonium Salt

Component d) is an alkylammonium salt comprising an anion of EDTA(“ethylenediamine tetraacetic acid” or “edetic acid”) or an anion of anEDTA analogue as described below, and at least one alkylammonium cationof Formula (I):

NR¹R²R³R^(4n+)  (I)

wherein each R¹-R⁴ is independently H, or a linear or branched C1-10group (as described herein), with the proviso that at least one of R¹-R⁴is not H.

Typically, and preferably, n=1. However, for ammonium salts containingmore than one nitrogen atom, such as ethylenediamine (NH₂CH₂CH₂NH₂ ⁺) itmay be possible for a mixture of +1 and +2 cations to exist (i.e.NH₂CH₂CH₂NH₃ ⁺ and NH₃CH₂CH₂NH₃ ²⁺). To a certain extent the formationof polycationic species may be prevented by providing an excess of theprecursor amine as described below. However, the person skilled in theart will appreciate when the formation of mixed cations is apossibility.

Each of R¹ to R⁴ may be the same or different, with the proviso that atleast one of R¹ to R⁴ is not H. Preferably all of the substituent groupsR¹ to R⁴ which are not H are the same. Preferred cations are thereforeNRH₃ ⁺, NR₂H₂ ⁺ and NR₃H⁺ or NR₄ ⁺ wherein the “R” groups in each arethe same. Primary, secondary and tertiary ammonium cations are preferredto quaternary cations as the former can be easily prepared by combiningthe appropriate amine with EDTA as described below.

Each of R¹ to R⁴ is independently H or a linear or branched C1-10 alkyl,alkenyl or alkynyl group, preferably C1-05. Most preferably each of R¹to R⁴ is a linear or branched C1-5 alkyl group, especially a linearC1-05 or C1-C3 alkyl group.

Each R¹ to R⁴ may independently be further substituted with one or moreOH or NH₂ (or NH₃ ⁺) groups. In an embodiment, for a substituent Rcontaining m carbon atoms, the substituent may contain a maximum of m−1OH and/or NH₂ groups per substituent. For instance, if R¹ is C8 then R¹may contain up to 7 OH groups, especially one OH unit attached to eachcarbon atom other than the carbon atom directly joined to the ammonium Natom. This embodiment is of particular relevance to the case in whichthe alkylammonium cation is derived from an aminopolyol (e.g. meglumine(MeNHCH₂(CHOH)₄CH₂OH)). As an alternative example, if R¹ is C3 then R¹may contain up to 2 OH groups, such as serinol (NH₂CH(CH₂OH)₂). In anembodiment at least one of R¹-R⁴ is a linear C1-C6 group substitutedwith at least one OH or NH₂ group.

In one embodiment any two of the groups R¹ to R⁴ taken together form aC4-C8, preferably C4-C6 ring, which may optionally contain one or moreexocyclic OH or NH₂ groups. If any two of the groups R¹ to R⁴ togetherform a ring then a single endocyclic O or NH unit may also be present.In particular, it is envisaged that morpholine salts may be used (i.e.if any two of R¹ to R⁴ together form a six-membered C4 ring containingone endocyclic 0 atom). In this embodiment two of the groups R¹ to R⁴along with N together form a morpholine ring, while the remaining groupsR¹ to R⁴ have the definition above.

Particularly preferred alkylammonium cations include those derived fromN-protonation, or in a less preferred embodiment N-alkylation, of anamine selected from:

Ethanolamine “ETA” (NH₂(CH₂CH₂OH));

Diethanolamine “DiETA” (NH(CH₂CH₂OH)₂);

meglumine (NH(CH₃)CH₂(CHOH)₄CH₂OH));

tris-hydroxymethylamine “TRIS” (N(CH₂OH)₃);

ethylenediamine (NH₂CH₂CH₂NH₂); or

serinol (NH₂CH(CH₂OH)₂).

It is preferred that the mass of the alkylammonium cation of Formula (I)is below 500 amu, preferably below 350, especially below 250 amu. Saltsof EDTA containing the ethanolammonium ion (HOCH₂CH₂NH₃ ⁺) areparticularly preferred in the invention. It is most preferred that theEDTA salt is a salt of EDTA with ethanolamine (ETA), preferably EDTAwith ETA only.

The alkylammonium cation is thought to aid in increasing the lipidsolubility the EDTA salt relative to a conventional metal (inorganic)EDTA salt such as disodium EDTA. As EDTA contains four carboxylic acidunits the alkylammonium salt may comprise up to four ammonium cationsand a tetraanionic EDTA anion.

As used herein, the term “EDTA” may represent ethylenediaminetetraaceticacid as such. Alternatively, EDTA as indicated herein may include bothethylenediaminetetraacetic acid itself and EDTA analogues. “EDTA” hereinthus includes “EDTA and analogues thereof” whenever context allows.Suitable EDTA analogues are those containing at least one glycinate unit(i.e. the unit —NCH₂COO—) within the molecule, preferably at least 2, atleast 3 or at least 4 glycinate units. Suitable EDTA analogues include:

Iminodiacetic acid (IDA)—(NH(CH₂CO₂H)₂;

Nitrilotriacetic acid (NTA)—N(CH₂CO₂H)₃;

Pentetic acid*—N(CH₂CO₂H)₂CH₂CH₂N(CH₂CO₂H)CH₂CH₂N(CH₂CO₂H)₂; * Alsoknown as “DTPA”

Egtazic acid—N(CH₂CO₂H)₂CH₂CH₂OCH₂CH₂OCH₂CH₂N(CH₂CO₂H)₂

NOTA—[N(CH₂CO₂H)CH₂CH₂]₃

DOTA—[N(CH₂CO₂H)CH₂CH₂]₄

In an embodiment the EDTA analogue has the structure indicated inFormula (II) below:

wherein n is 1-10, preferably 1-5, especially 1, 2 or 3;wherein X is CH₂, O or NR₄wherein R¹, R², R³ and R⁴ are each individually H or CH₂CO₂H, preferablyCH₂CO₂H; orwherein R¹ and R³ together represent a covalent bond (i.e. the EDTAanalogue is cyclic) and R² and R⁴ are each individually H or CH₂CO₂H,preferably CH₂CO₂H.

Amounts of EDTA and ratios of EDTA to (e) defined herein apply equallyto EDTA and EDTA analogues. In all embodiments it is preferred that EDTAis used as the counterion in component (d).

Formation of EDTA Salt

The EDTA salt may be pre-formed and dissolved or dispersed in one ormore of the components prior to forming the pre-formulation, or may beformed in situ. In situ formation is generally preferred for simplicityof operation. A suitable method for preparing the alkylammonium EDTAsalt involves dissolving EDTA (acid form) and the requisite alkylamine(base) in the solvent component (c), or in a solvent which is aprecursor to (or sub-component of) the solvent component (c), andproviding mixing until the solids are fully dissolved.

The inventors have established that as a general rule, for a mono-amineat least 3.0, preferably at least 3.5 (e.g. 3.5 to 10) molar equivalentsof amine (which is a precursor to the ammonium salt) are requiredrelative to the amount of EDTA in order to solubilize the salt in thesolvent component (c). As is described in the examples, the minimumratio between the amine and EDTA necessary to solubilize the salt variesdepending on the specific choice of alkylammonium salt. However, anappropriate molar ratio can be achieved by experimentation by simplyobserving at what molar excess of alkylamine the solid EDTA fullydissolves in the solvent. In an embodiment, a greater thanstoichiometric ratio of amine is added than is formally needed to formthe tetraamonium EDTA salt. For instance, as is described in thefollowing examples, efficient solubilisation of EDTA using TRIS mayrequire 5.0 or more equivalents of amine.

For certain di-amines or tri-amines the molar ratio to achieve adequateEDTA salt solubility may not be as high as for a mono-amine. Forpolyamines (diamines, triamines etc), such as NH₂CH₂CH₂NH₂, the requiredmolar ratio may be lower than that for a mono-amine. Suitable levels forpolyamines may be 2.0 or more (e.g. 2.0 to 4.0), or 2.5 or more. Again,suitable levels can be found by optimisation. As a guide, the molarequivalents of amine discussed above may represent the molar ratio ofmono-amine to EDTA or the ratio of amine moieties to EDTA where theamine (or mixture of amines) has more than one amine moiety in themolecule (either individually or on average for a mixture).

There is no upper limit on the number of equivalents of amine which maybe present, although it will be appreciated that typically no more amineshould be included than is necessary to ensure efficient solubilisation.A typical practical limit may be 20 equivalents, preferably 10equivalents.

The inventors have established that in order to form the alkylammoniumEDTA salt, it is necessary to begin with the acid form of EDTA ratherthan the commonly used disodium EDTA (EDTA(Na)). Neither EDTA (edeticacid) nor EDTA(Na) are soluble in suitable/preferred solvents (e.g.EtOH/PG) without an alkylamine (e.g. ETA), even after several months ofmixing. Surprisingly, EDTA(Na) is insoluble in EtOH/PG even in thepresence of ETA.

A typical procedure for producing the salt therefore involves dissolvingthe free tetraacid EDTA (which may be a hydrate) in the solvent (c), orin a solvent which is a precursor to the solvent component (c), whichcomprises at least a mono-alcoholic solvent such as ethanol, and mayalso comprise a polar co-solvent as previously described, preferably ina mixture of ethanol and PG. The requisite number of equivalents ofalkylamine are then added and the mixture is agitated, e.g. byend-over-end rotation or magnetic stirring, until the EDTA is dissolved,as can be established by visual observation. 24 h of mixing is usuallyadequate to ensure efficient solubility, e.g. in the case of theformation of an ETA/EDTA salt.

It is also within the scope of the invention to form the salt in asolvent which is a precursor to the solvent component (c). By“precursor” it is meant that the solvent in which the EDTA salt isformed is not identical to the final composition of solvent component(c), but that the content of solvent(s) in the precursor can be adjustedto arrive at the final composition of the solvent (c) in thepre-formulation. As an example, the salt may be formed in a mixture ofEtOH:PG (1:2) and additional ethanol added during or after saltformation in order to reach a final composition of EtOH:PG (1:1) forcomponent (c).

Ratio of Alkylamine to EDTA

The inventors have surprisingly established that above a certain ratioof alkylamine:EDTA the chemical stability of the active agent in thepre-formulation begins to decrease. This may be a result of reactionbetween the excess alkylamine and the active agent, either directly orvia degradation products. Accordingly, it is preferred that the amountof alkylamine chosen is sufficient to fully solubilize all of the EDTAin the solvent component (c) but is not significantly beyond this level.It is preferred that the amount of alkylamine included is no more than 2times the required level to achieve complete solubility, preferably nomore than 1.5 times, preferably no more than 1.2 times. The amount ofalkylamine necessary to fully solubilize ETDA in the solvent component(c) can be established by the methods described previously.

In an embodiment component (d) comprises an alkylammonium counterionhaving only one amino or alkylamino group and the ratio of EDTA:thetotal of said alkylammonium counterion and any amine free base thereofin the pre-formulation is 1:≥3.0; preferably 1:≥3.5, most preferably inthe range of 1:3.0 to 1:10.

In an embodiment component (d) comprises an alkylammonium counterionhaving two or more amino and/or alkylamino groups, wherein the ratio ofEDTA:the total of said alkylammonium counterion and any amine free basethereof in the pre-formulation is 1:≥2.0; preferably in the range of1:2.0 to 1:4.0.

In a particularly preferred aspect the EDTA salt is an ETA salt of EDTA.The inventors have established that in this embodiment in order to fullysolubilise EDTA in the solvent component (c) (e.g. a mixture of EtOH/PG50:50) it is necessary to include around at least 3.5 molar equivalentsof ETA relative to the amount of EDTA. Accordingly, the amount of ETA toEDTA is preferably no more than 7:1. The equivalents of ETA to EDTA arepreferably in the range of 3.5 to 7 (mol/mol), preferably 3.5 to 5, mostpreferably 3.5 to 4.5. Most preferably 4 equivalents of ETA are usedrelative to the amount of EDTA (mol/mol).

Amount of EDTA Salt

The level of alkylammonium EDTA salt is chosen to ensure appropriatestability of the components of the lipid vehicle and active agent forthe storage duration required and under the chosen storage conditions.Factors to be considered when determining appropriate amounts ofalkylammonium EDTA salt include: the reactivity of the lipid componentsand active agent, the loading of active agent, the molecular mass of theactive agent, storage conditions (oxygen content, humidity,temperature), the duration of oxidative protection required and theconcentration of metal ions present in the pre-formulation (which maycatalyse decomposition processes).

In order to suppress the catalytic activity of metals, e.g. Fe, thepre-formulation will typically include the EDTA salt at a level suchthat the ratio of EDTA salt to metal (e.g Fe, especially in the form ofFe(II) and Fe(III) ions) is at least around 2:1 (mol/mol), i.e. the EDTAsalt is present in at least a 2 times molar excess. In a typicalprocedure the molar ratio will be based upon the maximum estimated metalion (especially Fe ion) concentration and EDTA provided in a ratio ofaround 2:1 to this maximum estimate. The result in practice will then be2:1 or greater molar ratio of EDTA to metal (e.g. Fe) ions.

The inventors have established that there is a preferred level of EDTA,above which no advantage in terms of oxidation resistance of theformulation is observed, and indeed the stability may be somewhatreduced. This is influenced by the amount of metal ions (e.g. Fe ions)present in the formulation as is discussed in detail in the“Experimental” section. However, in general a suitable amount of EDTAsalt in the pre-formulation (calculated in terms of EDTA free acid) willbe 0.001-0.02 wt % (10-200 ppm), preferably 0.001-0.015 wt % (10-150ppm), especially 0.002-0.015 wt % (20-150 ppm). A particularly preferredlevel is 0.005-0.015 wt. % (50-150 ppm), most preferably 0.008-0.012 wt.% (80-120 ppm). A level of 100 ppm is suitable for protecting against upto 10 ppm of metal (iron equivalents) which is reasonable for ensuringappropriate drug product robustness.

In certain embodiments, the levels of EDTA (based on the weight of EDTAalone and not including the amine countercations) may range from 0.001to 0.8 wt % (10 to 8000 ppm), 0.002 to 0.5 wt % (20 to 5000 ppm), 0.005to 0.2 wt % (50 to 2000 ppm) or 0.01 to 0.1 wt % (100 to 1000 ppm) ofthe pre-formulation. In certain embodiments the level of EDTA may rangefrom 0.001 to 0.050 wt % of the formulation (10 to 500 ppm), preferably0.02 to 0.30 wt % (20 to 300 ppm) of the formulation.

The level of alkylamine to be added can be established once the optimumratio of alkylamine to EDTA is found, as described in precedingsections.

In an embodiment the ratio of (d) to (e) is in the range 1:1 to 1:5000(w/w), preferably 1:1 to 1:500 (w/w), preferably in the range of 1:50 to1:300.

Water Content

The inclusion of EDTA salts containing an alkylammonium ion of Formula(I) allows for an antioxidant to be included in the pre-formulation atlow levels of water. It is however extremely difficult to completelyeliminate all traces of water (especially from the raw materials). Evenif essentially water-free formulations could be achieved,pre-formulations will typically be stored in ready-to-use form, e.g. insyringes and possibly under refrigerated conditions. Syringes are oftennot completely air-tight meaning that the level of water in thepre-formulation may increase to an appreciable level over time, e.g.over months, even if the initial level of water is insignificant.

The initial absolute level of water in the pre-formulation is between 0to 1.0 wt. %. Preferably the water content is less than 1.0 wt. %,preferably less than 0.8 wt %, preferably less than 0.5 wt %. Mostpreferably, the level of water is in the range of 0.1 to 0.9 wt. %,especially 0.2 to 0.8 wt. %. These levels refer to the absolute level ofwater and not added levels of water. Any unavoidable trace of waterpresent within components a), b) or c) is included in this stated levelof water. After 3 months of storage, the absolute water level ispreferably no more than 1.5 wt %. Absolute levels of water can bemeasured by methods well known in the art such as Karl Fischertitration. In particular, the water content is preferably measuredaccording to the procedure in United States Pharmacopoeia (USP 40—NF 35,USP <921> Water determination, Method Ia.

Component e)—Active Agent

The pre-formulations of the present invention contain one or moresomatostatin receptor agonist(s), equivalently referred to herein as“active agents”. The somatostatin receptor agonist(s) may be endogenousor may be synthetic analogues.

Somatostatin receptor agonists often have a short residence time in thebody due to rapid breakdown (in vivo degradation) or excretion. Byadministering such agents in the form of a depot composition formed fromthe pre-formulation of the present invention, the agents are provided ata sustained level for a length of time which may stretch to days, weeksor even several months in spite of having rapid clearance rates. Thisoffers obvious advantages in terms of stability and patient complianceover dosing multiple times each day for the same period. In onepreferred embodiment, the somatostatin receptor agonist has a biologicalhalf life (upon entry into the blood stream) of less than 1 day,preferably less than 12 hours and more preferably less than 6 hours. Insome cases this may be as low as 1-3 hours or less.

The amount of somatostatin receptor agonist to be formulated with thepre-formulations of the present invention will depend upon thefunctional dose and the period during which the depot composition formedupon administration is to provide sustained release. Typically, the doseformulated for a particular agent will be around the equivalent of thenormal daily dose multiplied by the number of days the formulation is toprovide release. Evidently this amount will need to be tailored to takeinto account any differences in bioavailability or adverse effects of alarge dose at the beginning of treatment and so this will generally bethe maximum dose used. The precise amount suitable in any case willreadily be determined by suitable experimentation.

A further considerable advantage of the depot compositions of thepresent invention is that the somatostatin receptor agonist(s) withinthe depot formed by the pre-formulation upon administration are releasedgradually over long periods without the need for repeated dosing. Thepre-formulations are thus highly suitable for situations where patientcompliance is difficult, unreliable or where a level dosage is highlyimportant, such as mood-altering actives, those actives with a narrowtherapeutic window, and those administered to children or to peoplewhose lifestyle is incompatible with a reliable dosing regime and for“lifestyle” actives where the inconvenience of repeated dosing mightoutweigh the benefit of the active.

The active agent component e) is a somatostatin receptor agonist ormixture of somatostatin receptor agonists. Where reference is made to“somatostatin receptor agonist”, it is to be understood that this alsoencompasses mixtures of somatostatin receptor agonists unless contextpermits otherwise.

Somatostatins (Growth Hormone Release Inhibiting Factors, SSTs) arenatural peptide hormones with a wide distribution in animals, acting asneurotransmitters in the central nervous system, and having diverseparacrine/autocrine regulatory effects on several tissues. Twobiologically active products are known in higher species, SST-14 andSST-28, the latter being a congener of SST-14 extended at theN-terminus.

SST-14 is a 14 residue cyclic peptide hormone having the sequenceAla-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys, where the twocysteine residues are connected by a disulphide bridge to generate atype II (3-turn at the key binding sequence of Phe-Trp-Lys-Thr. Thebiological half-life of natural SST-14 is very short (1-3 minutes) andso is in itself a challenging peptide for therapeutic use, but anincreasing number of somatostatin receptor agonists are becomingavailable with higher activities and/or longer clearance times in vivo.

Somatostatin receptor agonists (SRAs), such as SST-14, SST-28,octreotide, lanreotide, vapreotide, pasireotide (SOM 230) and relatedpeptides, are used or indicated in the treatment of a variety ofconditions where they are typically administered over an extendedperiod. These agonists form a preferred class of somatostatin receptoragonists for use as component e) in the present invention. Octreotide,for example, is the synthetic octapeptide with sequenceD-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (2-7 disulphide bridge) and istypically administered as an acetate salt. This SST-14 derivativeretains the key Phe-(D)Trp-Lys-Thr (3-turn required for in vivo SST-likeactivity but, in contrast to the natural hormone, has a terminalhalf-life of around 1.7 hours. Octreotide is used in treatment ofconditions including carcinoid tumours and acromegaly, and is typicallyadministered over a sustained period of weeks, or more commonly manymonths or years. Somatostatin receptor agonists are of particularinterest for the treatment of many different types of cancers since awide variety of tumours are found to express somatostatin receptors(SSTRs). There are five known types of SSTRs (SSTR1-SSTRS), showingequally high affinity for SST-14. The most investigated somatostatinreceptor agonists, including octreotide, show high selectivity for SSTR2and SSTRS; thus, octreotide is of particular interest for the treatmentof tumours expressing these types of receptors.

The most common “simple” formulation of Octreotide is “Sandostatin”®from Novartis. This is an aqueous solution for subcutaneous (s.c)injection, and a 100 μg dose reaches a peak concentration of 5.2 ng/mlat 0.4 hours post injection. The duration of action can be up to 12hours but s.c. dosing is generally carried out every 8 hours. Evidently,s.c. injection 3 times daily for periods of months or years is not anideal dosing regime.

Following a single subcutaneous dose of pasireotide, human plasma levelstypically peak quickly, at around 15 minutes to 1 hour after dosing,with an initial half-life of 2-3 hours following that peak. Althoughclearance half-life is greater for later phases of the decline, it isclear that the Cmax/Cave for such a delivery will be rather high.

Pasireotide LAR is a long acting formulation of pasireotide whichaddresses some of the above issues. However, this is a polymermicroparticle based system with the inherent limitations of such asystem, as are known in the art and described herein above.

Carcinoid tumours are intestinal tumours arising from specialised cellswith paracrine functions (APUD cells). The primary tumour is commonly inthe appendix, where it is clinically benign. Secondary, metastatic,intestinal carcinoid tumours secrete excessive amounts of vasoactivesubstances, including serotonin, bradykinin, histamine, prostaglandins,and polypeptide hormones. The clinical result is carcinoid syndrome (asyndrome of episodic cutaneous flushing, cyanosis, abdominal cramps, anddiarrhea in a patient with valvular heart disease and, less commonly,asthma and arthropathy). These tumours may grow anywhere in thegastrointestinal tract (and in the lungs) with approximately 90% in theappendix. The remainder occurs in the ileum, stomach, colon or rectum.Currently, treatment of carcinoid syndrome starts with i.v. bolusinjection followed by i.v. infusion. When sufficient effect on symptomshas been established, treatment with a depot formulation of octreotideformulated in ploy lactic-co-glycolic acid (PLGA) microspheres isstarted. However, during the first two weeks or more after injection ofthe depot, daily s.c. injections with octreotide are recommended tocompensate for the slow release from the PLGA spheres.

Since SST-14 is a peptide hormone, typical somatostatin receptoragonists will be peptides, especially of 14 or fewer amino acids.Preferably such peptides will be structurally constrained such as bybeing cyclic and/or having at least one intramolecular cross-link.Amide, ester or particularly disulphide crosslinks are highly suitable.Preferred constrained peptides will exhibit a type-2 β turn. Such a turnis present in the key region of somatostatin. Peptides may contain onlyamino acids selected from those 20 α-amino acids indicated in thegenetic code, or more preferably may contain their isomers and othernatural and non-natural amino acids, (generally α, μ or γ, L- or D-aminoacids) and their analogues and derivatives. The term “somatostatinreceptor agonist” as used herein may optionally also encompass SST-14and/or SST-28, since these are viable peptide actives when formulated assalts in the very high performance slow-release formulations describedherein.

Amino acid derivatives and amino acids not normally used for proteinsynthesis are especially useful at the termini of the somatostatinreceptor agonist, where the terminal amino or carboxylate group may besubstituted by or with any other functional group such as hydroxy,alkoxy, ester, amide, thio, amino, alkyl amino, di- or tri-alkyl amino,alkyl (by which is meant, herein throughout C₁-C₁₈ alkyl, preferablyC₁-C₈ alkyl e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-, sec-or t-butyl etc.), aryl (e.g phenyl, benzyl, napthyl etc) or otherfunctional groups, preferably with at least one heteroatom andpreferably having no more than 10 atoms in total, more preferably nomore than 6.

Particularly preferred somatostatin receptor agonists are constrainedpeptides of 6 to 10 α-amino acids, of which particular examples includeoctreotide, lanreotide (of sequenceNH₂-(D)Naph-Cys-Tyr-(D)Trp-Lys-Val-Cys-Thr-CONH₂ and its cyclicderivative of sequence NH₂-(D)Naph-Cys-Tyr-(D)Phe-Lys-Val-Cys-Thr-CONH₂both having a Cys-Cys intramolecular disulphide crosslink), pasireotide(aka SOM 230) and vapreotide. Most preferred are octreotide andpasireotide.

The somatostatin receptor agonist will generally be formulated as 0.1 to12% by weight of the total formulation (based on the amount of freebase). Typical values will be 0.1 to 10%, 0.5 to 9%, preferably 1 to 9%,and in some embodiments 1 to 8% or 1 to 7%. A somatostatin receptoragonist content of 2-6% is suitable in certain embodiments.

The somatostatin receptor agonist will generally be formulated in anamount of 5 to 100 mg per mL of pre-formulation, preferably 10 to 80mg/mL. In some embodiments, particularly where the somatostatin receptoragonist comprises, consists essentially of or consists of octreotide,the level of octreotide may be 10 to 50 mg/mL, such as 10 to 30 mg/mL(e.g. 10 mg/mL, 20 mg/mL, 30 mg/mL), with a level of 15 to 25 mg/mL(e.g. 20 mg/mL) being particularly appropriate. Where the somatostatinreceptor agonist comprises, consists essentially of or consists ofpasireotide, the level of pasireotide may be 10 to 100 mg/mL, such as 20to 60 mg/mL (e.g. 20 mg/mL, 40 mg/mL, 60 mg/mL).

Doses of the somatostatin receptor agonist suitable for inclusion in theformulation, and thus the volume of formulation used, will depend uponthe release rate (as controlled, for example by the solvent type andamount use) and release duration, as well as the desired therapeuticlevel, the activity and the rate of clearance of the particular activechosen. Typically an amount of 1 to 500 mg per dose would be suitablefor providing a therapeutic level for between 7 and 90 days. This willpreferably be 5 to 300 mg. For octreotide, the level will typically bearound 10 to 180 mg (e.g. for a 30 to 90 day duration). Preferably, theamount of octreotide will be around 0.2 to 3 mg per day betweeninjections. Thus a depot administered every 30 days would have 6 to 90mg or a 90 day depot have 18 to 270 mg of octreotide.

For Pasireotide, the dosage would typically be an amount of around 0.05to 40 mg per week of depot duration, preferably 0.1 to 20 mg per weekduration (e.g. 1 to 5 mg per week) for a duration of 1 to 24 weeks,preferably 2 to 16 (e.g. 3, 4, 8, 10 or 12) weeks. In an alternativeembodiment the pre-formulation may be formulated for dosing weekly (e.g.every 7±1 days). A total dose of 0.05 to 250 mg of Pasireotide per dosewould be suitable for providing a therapeutic level for between 7 and168 days. This will preferably be 0.1 to 200 mg, e.g. 0.2 to 150 mg, 0.1to 100 mg, 20 to 160 mg etc. Evidently, the stability of the active andeffects on the release rate will mean that the loading to duration maynot be a linear relationship. A depot administered every 30 days mighthave, for example 0.2 to 20 mg of Pasireotide, or a 90 day depot mighthave 30 to 60 mg of Pasireotide.

Where the salt of said peptide somatostatin receptor agonist is used inthe formulations of the present invention, this will be a biologicallytolerable salt. Suitable salts include the acetate, pamoate, chloride orbromide salts. The chloride salt is most preferred.

In a further preferred embodiment of the invention the pre-formulationcomprising: a lipid controlled-release matrix comprising:

-   -   a) at least one di-acyl lipid;    -   b) at least one phospholipid;    -   c) at least one biocompatible, organic solvent;    -   d) an alkyl ammonium EDTA salt; and    -   e) at least one somatostatin receptor agonist;        wherein the pre-formulation has a water content in the range of        0 to 1.0 wt %.

In a further preferred embodiment of the invention the pre-formulationcomprises:

-   -   a) at least one di-acyl lipid;    -   b) at least one phospholipid;    -   c) at least one biocompatible, organic solvent;    -   d) an alkyl ammonium EDTA salt; and    -   e) octreotide or a salt thereof.

In a further preferred embodiment of the invention the pre-formulationcomprises:

-   -   a) GDO;    -   b) PC;    -   c) ethanol and PG;    -   d) an alkyl ammonium EDTA salt; and    -   e) octreotide or a salt thereof.

In a further preferred embodiment of the invention the pre-formulationcomprises:

-   -   a) 33-43% of GDO;    -   b) 33-55% of PC;    -   c) 8-18% of a mixture of ethanol and PG;    -   d) 10-200 ppm of an alkyl ammonium EDTA salt, preferably and ETA        salt of EDTA; and    -   e) 0.1-12 wt % of octreotide or a salt thereof, preferably        octreotide chloride.

In a further preferred embodiment of the invention the pre-formulationcomprises:

-   -   a) 33-60 wt % GDO;    -   b) 33-55 wt % SPC;    -   c) 2-20 wt % EtOH and PG;    -   d) 0.001-0.050 wt % EDTA;    -   e) 1-10 wt % OCT(Cl);    -   based on the total weight of the pre-formulation.

In a further preferred embodiment of the invention the pre-formulationcomprises:

-   -   a) 33-43 wt % GDO;    -   b) 33-55 wt % SPC;    -   c) 8-18 wt % EtOH and PG;    -   d) 0.001-0.020 wt % EDTA;    -   e) 0.1-12 wt % OCT(Cl);

based on the total weight of the pre-formulation.

In a further preferred embodiment of the invention the pre-formulationcomprises:

-   -   a) 42.3 wt % GDO;    -   b) 42.3 wt % SPC;    -   c) 6.5 wt % EtOH and 6.5 wt % PG;    -   d) 0.010 wt % EDTA and 0.008 wt % ETA;    -   e) 2.27 wt % OCT(Cl);    -   based on the total weight of the pre-formulation;    -   and wherein the wt % values may vary with +/−20%, preferably        +/−10%, more preferably +/−5% of said wt % values.

The term “pre-formulation” herein is a pharmaceutical composition,preferably is a parenteral pharmaceutical composition, more preferablyis an injectable parenteral pharmaceutical composition, even morepreferably is an injectable parenteral pharmaceutical composition forsubcutaneous or intra-muscular application, even more preferably is aninjectable parenteral pharmaceutical composition for subcutaneousapplication.

Optional Additional Components

In one particularly preferred embodiment of the present invention, thecompositions (pre-formulations and resulting depots) do not includefragmentation agents, such as polyethyleneoxide or poly(ethylene glycol)(PEG) fragmentation agent, e.g. a PEG grafted lipid and/or surfactant.

For example, the compositions preferably do not include fragmentationagents such as Polysorbate 80 (P80), or other Polysorbates (e.g.Polysorbate 20), PEGylated phospholipids (PEG-lipids such asDSPE-PEG(2000), DSPE-PEG(5000), DOPE-PEG(2000) and DOPE-PEG(5000)),Solutol HS 15, PEGylated fatty acids (e.g. PEG-oleate), blockco-polymers such as Pluronic® F127 and Pluronic® F68, ethoxylated castoroil derivatives (e.g. Chremophores), PEGylated glyceryl fatty acidesters (such as TMGO-15 from Nikko Chemicals) and PEGylated tocopherols(such as d-alpha tocopheryl poly(ethylene glycol)1000 succinate known asVitamin E TPGS from Eastman.

However, the active agent as a powder (e.g. in the kit of theinvention), as well as active agent dissolved in the lipid formulation,may gain stability (both storage and in vivo stability) by certainstabilising additives. Such additives include sugars (e.g. sucrose,trehalose, lactose etc.), polymers (e.g. polyols such as carboxy methylcellulose), amino acids (such as methionine, glutamate, lysine etc.),lipid-soluble acid components such as HCl, anionic lipids and/or surfaceactive agents (such as dioleoyl phosphatidyl glycerol (DOPG),palmitoyloleoyl phosphatidylglycerol (POPG) and oleic acid (OA)).

Single-dose formats must remain stable and potent in storage prior touse, but are disposable after the single use. It is a remarkable findingthat non-aqueous pre-formulations comprising an alkylammonium EDTA salthave enhanced storage stability at elevated temperatures, such as at 25°C. or even 40° C. This offers advantages in terms of ease oftransportation and storage (no need for refrigeration). It is preferredthat a single dose format has a stability such that after storage for 2months at 25° C. (with air in head space), the assayed active agentconcentration is at least 95% that of the initial assayed active agentconcentration, and after 3 months, the assayed active agentconcentration is at least 90% that of the initial assayed active agentconcentration.

It is preferred that a single dose format has a stability such thatafter storage for 2 months at 40° C. (with air in head space), theassayed active agent concentration is at least 85% that of the initialassayed active agent concentration, and after 3 months, the assayedactive agent concentration is at least 80% that of the initial assayedactive agent concentration.

Multi-dose formats must not only remain stable and potent in storageprior to use, but must also remain stable, potent andrelatively/effectively free of bacteria over the multiple-dose useregimen administration period after the first use in which a seal hasbeen compromised. For this reason multi-dose formats often require anantimicrobial or microbial-static agent, e.g. bacteriostatic agent,preservative.

However, the production of preserved pharmaceutical preparationscontaining protein or peptide actives has often proven difficult, aswhen preservatives are used, these give rise to stability problems.Often the proteins are inactivated and aggregates are formed, which maysometimes lead to reported injection site intolerance or immunogenicityto the active. This can be further aggravated by additional excipientsor formulation components.

In one aspect each of the embodiments herein can optionally contain anantimicrobial or microbial-static agent, which includes bacteriostaticagents and preservative. Such agents include benzalkonium chloride,m-cresol, benzyl alcohol or other phenolic preservatives. Typicalconcentrations as known in the art can be used.

Additional components above those mentioned as components a) to e) will,where present at all, preferably be present in an amount of 0 to 5%(e.g. 0.01% to 5%) by weight, preferably no more than 2% by weight andmore preferably no more than 1% by weight.

In one embodiment, components a) and b) (allowing for any impurityinherent in the nature of these components) make up at least 95% of thelipid components of the composition. Preferably at least 99% of thetotal lipid content of the pre-formulation consists of components a) andb). Preferably the lipid component of the pre-formulation consistsessentially of components a) and b).

Administration

The pre-formulations of the present invention are generally formulatedto be administered parenterally. This administration will generally notbe an intra-vascular method but will preferably be subcutaneous (s.c.),intracavitary or intramuscular (i.m.). Typically the administration willbe by injection, which term is used herein to indicate any method inwhich the formulation is passed through the skin, such as by needle,catheter or needle-less (needle-free) injector. Preferred parenteraladministration is by i.m or s.c. injection, most preferably by deep s.c.injection. An important feature of the composition of the invention isthat it can be administered both by i.m. and s.c. and other routeswithout toxicity or significant local effects. It is also suitable forintracavital administration. The deep s.c. injection has the advantageof being less deep and less painful to the subject than the (deep) i.m.injection used for some current depots and is technically most suitablein the present case as it combines ease of injection with low risk oflocal side effects. It is a surprising observation of the presentinventors that the formulations provide sustained release of activeagent over a predictable time period by both subcutaneous andintramuscular injection. This therefore allows the site of injection tobe varied widely and allows the dose to be administered without detailedconsideration of the tissue depth at the site of injection.

In one embodiment the lipid pre-formulations of the present inventionprovide non-lamellar liquid crystalline depot compositions upon exposureto aqueous fluids, especially in vivo. As used herein, the term“non-lamellar” is used to indicate a normal or reversed liquidcrystalline phase (such as a cubic or hexagonal phase) or the L₃ phaseor any combination thereof. The term liquid crystalline indicates allhexagonal, all cubic liquid crystalline phases and/or all mixturesthereof. Hexagonal as used herein indicates “normal” or “reversed”hexagonal (preferably reversed) and “cubic” indicates any cubic liquidcrystalline phase unless specified otherwise. The skilled reader willhave no difficulty in identifying those compositions having appropriatephase behaviour by reference to the description and Examples providedherein, and to WO2005/117830, but the most favoured compositional areafor phase behaviour is where ratio of components a:b are in the regionof 40:60 to 70:30, preferably 45:55 to 55:45 and more preferably 40:60to 54:46. Ratios of around 50:50 (e.g. 49:51 to 51:49) are highlypreferred, most preferably around 50:50.

It is important to appreciate that the pre-formulations of the presentinvention are of low viscosity. As a result, these pre-formulations mustnot be in any bulk liquid crystalline phase since all liquid crystallinephases have a viscosity significantly higher than could be administeredby syringe or similar injecting dispenser. The pre-formulations of thepresent invention will thus be in a non-liquid crystalline state, suchas a solution, L₂ or L₃ phase, particularly solution or L₂. The L₂ phaseas used herein throughout is preferably a “swollen” L₂ phase containinggreater than 5 wt %, preferably greater than 7%, and most preferablygreater than 9% of organic mono-alcoholic solvent (component c) having aviscosity reducing effect.

The pre-formulations described herein are preferably of “low viscosity”.This may be indicated, for example by the ability to be dispensed from a1 ml disposable syringe through a small gauge needle. Preferably, thelow viscosity mixtures can be dispensed through a needle of 19 awg,preferably smaller than 19 gauge, more preferably 23 awg (or mostpreferably even 27 gauge) needle by manual pressure. In a particularlypreferred embodiment, the low viscosity mixture should be a mixturecapable of passing through a standard sterile filtration membrane suchas a 0.22 μm syringe filter. A typical range of suitable viscosities forthe pre-formulations of the invention would be, for example, 1 to 1000mPas, preferably 10 to 800 mPas, more preferably 50 to 750 mPas and mostpreferably 50 to 600 mPas at 20° C.

Upon administration, many of the preferred lipid-based pre-formulationsof the present invention undergo a phase structure transition from a lowviscosity mixture to a high viscosity (generally tissue adherent) depotcomposition. Generally this will be a transition from a molecularmixture, swollen L₂ and/or L₃ phase to one or more (high viscosity)liquid crystalline phases such as normal or reversed hexagonal or cubicliquid crystalline phases or mixtures thereof. Further phase transitionsmay also take place following administration. Obviously, complete phasetransition is not necessary for the functioning of the invention but atleast a surface layer of the administered mixture will form a liquidcrystalline structure. Generally this transition will be rapid for atleast the surface region of the administered formulation (that part indirect contact with air, body surfaces and/or body fluids). This willmost preferably be over a few seconds or minutes (e.g. from 1 second upto 30 minutes, preferably up to 10 minutes, more preferably 5 minutes ofless). The remainder of the composition may change phase to a liquidcrystalline phase more slowly by diffusion and/or as the surface regiondisperses.

Without being bound by theory, it is believed that upon exposure toexcess aqueous fluid, the pre-formulations of the invention lose some orall of the organic solvent included therein (e.g. by diffusion) and takein aqueous fluid from the bodily environment (e.g. the in vivoenvironment). For certain lipid pre-formulations as described herein, atleast a part of the formulation preferably generates a non-lamellar,particularly liquid crystalline phase structure. In most cases thesenon-lamellar structures are highly viscous and are not easily dissolvedor dispersed into the in vivo environment. The result is a monolithic“depot” generated in vivo with only a limited area of exposure to bodyfluids. Furthermore, because the non-lamellar structure has large polar,apolar and boundary regions, the lipid depot is highly effective insolubilising and stabilising active agents such as peptides andprotecting these from degradation mechanisms. As the depot compositionformed from the pre-formulation gradually degrades over a period ofdays, weeks or months, the active agent is gradually released and/ordiffuses out from the composition. Since the environment within thedepot composition is relatively protected, the pre-formulations of theinvention are highly suitable for active agents with a relatively lowbiological half-life (see above).

By incorporation of a co-solvent into the pre-formulations, as describedfor the first time in WO2012/160213, it is believed that the rate ofphase transition to a non-lamellar (e.g. liquid crystalline) phase atthe surface of the injected pre-formulation can be enhanced incomparison with compositions containing organic solvents in thesubstantial absence of water. The performance of the resulting depot isthus improved and further control over the release of active agentachieved.

The depot systems formed by the formulations of the present inventionare highly effective in protecting the active agent from degradation andthus allow an extended release period. The formulations of the inventionthus may provide in vivo depots of peptide active agents which requireadministration only once every 5 to 90 days preferably 5 to 60 days,more preferably 6 to 32. Evidently, a longer stable release period isdesirable for patient comfort and compliance, as well as demanding lesstime from health professionals if the composition is not to beself-administered. Where the composition is to be self-administered,patient compliance may be aided by a weekly (e.g. every 7 days,optionally ±1 day), bi-seekly (e.g. every 14 days, optionally ±2 days)or monthly (e.g. every 28 or 30 days (optionally ±7 days) administrationso that the need to administer is not forgotten.

A considerable advantage of the depot precursors of the presentinvention is that they are stable homogeneous phases. That is to say,they may be stored for considerable periods (preferably at least 6months) at room or refrigerator temperature, without phase separation.As well as providing advantageous storage and facile administration,this allows for the dose of somatostatin receptor agonist to be selectedby reference to the species, age, sex, weight, and/or physical conditionof the individual subject, by means of injecting a selected volume.

The present invention thus provides for methods comprising the selectionof a dosing amount specific to an individual, particularly by subjectweight. The means for this dose selection is the choice ofadministration volume.

In one preferred aspect, the present invention provides apre-formulation comprising components a), b), c), d), and e), and 0-1.0%water. The amounts of these components will typically be in the range20-60% a), 20-60% b), 1-30% c) and 0.001-0.8% d).

The pre-formulations of the present invention are highly advantageous inthat they are stable to prolonged storage in their final “administrationready” form. As a result, they may readily be supplied foradministration either by health professionals or by patients or theircarers, who need not be fully trained health professionals and may nothave the experience or skills to make up complex preparations. This isparticularly important in long-duration, slow-effecting diseases such asdiabetes.

Devices

In a yet further aspect, the present invention provides a disposableadministration device (which is also to include a device component)pre-loaded with a measured dose of a pre-formulation of the presentinvention. Such a device will typically contain a single dose ready foradministration, and will generally be sterile-packed such that thecomposition is stored within the device until administration. Suitabledevices include cartridges, ampoules and particularly syringes andsyringe barrels, either with integral needles or with standard (e.g.luer) fittings adapted to take a suitable disposable needle.

For a device comprising a single dose of a pre-formulation comprisingoctreotide, an amount of octreotide of 10 to 180 mg would be suitablefor providing a therapeutic level for between 7 and 90 days.

Preferably, the amount of octreotide will be around 0.2 to 3 mg per daybetween injections. Thus a depot administered every 30 days would have 6to 90 mg or a 90 day depot have 18 to 270 mg of octreotide.

For Pasireotide, the dosage would typically be an amount of around 0.05to 40 mg per week of depot duration, preferably 0.1 to 20 mg per weekduration (e.g. 1 to 5 mg per week) for a duration of 1 to 24 weeks,preferably 2 to 16 (e.g. 3, 4, 8, 10 or 12) weeks. In an alternativeembodiment the pre-formulation may be formulated for dosing weekly (e.g.every 7±1 days). A total dose of 0.05 to 250 mg of Pasireotide per dosewould be suitable for providing a therapeutic level for between 7 and168 days. This will preferably be 0.1 to 200 mg, e.g. 0.2 to 150 mg, 0.1to 100 mg, 20 to 160 mg etc. Evidently, the stability of the active andeffects on the release rate will mean that the loading to duration maynot be a linear relationship. A depot administered every 30 days mighthave, for example 0.2 to 20 mg of Pasireotide, or a 90 day depot mighthave 30 to 60 mg of Pasireotide.

Kits

The pre-filled devices of the invention may also suitably be included inan administration kit, which kit also forms a further aspect of theinvention. In a still further aspect, the invention thus provides a kitfor the administration of at least one somatostatin receptor agonist,said kit containing a measured dose of a formulation of the inventionand optionally an administration device or component thereof. Preferablythe dose will be held within the device or component, which will besuitable for i.m. or preferably s.c. administration. The kits mayinclude additional administration components such as needles, swabs,etc. and will optionally and preferably contain instructions foradministration. Such instructions will typically relate toadministration by a route as described herein and/or for the treatmentof a disease indicated herein above.

The invention provides for a pre-filled administration device asindicated herein and a kit as indicated herein comprising apre-formulation as described herein.

In an alternative aspect of the present invention, the “kit” may containat least two vessels, a first containing a low viscosity mixture ofcomponents a) to d), as described here, and a second containing ameasured dose of at least one active agent as described herein.

Such a “two component kit” may comprise the active agent as a powderformulation in one vial or pre-filled syringe and components a) to d) ina second vial or pre-filled syringe. In the case of two syringes, beforeinjection, the pre-filled syringes are connected and the powdercomprising active agent is mixed with the matrix formulation by movingthe syringe barrels back and forth, forming a solution or suspensionwhich is injected. Alternatively, the liquid lipid formulation is drawnfrom one vial, or is pre-filled into a syringe, and is injected into avial containing powdered active agent (e.g. peptide). This formulationmay subsequently be mixed by hand shaking or other suitablereconstitution method (e.g. vortex mixing etc.).

The solvent component may be present in either or both vessels (e.g.vials or syringes). Where the solvent is at least partially constitutedwith the active agent, this will generally be in the form of a solutionor suspension.

In this aspect, the invention therefore provides a two component kitcomprising

i) a first vessel containing a low viscosity mixture of components a) toc) as described herein;

ii) an optional second vessel containing at least one peptide activeagent,

iii) an antioxidant component d) optionally in a third vessel,preferably in the second vessel, or most preferably in the first vessel;

iv) optionally and preferably at least one of:

-   -   1) at least one syringe (which may be one or both of said first        and second vessels);    -   2) a needle for administration, such as those described herein;    -   3) instructions for generation of a composition of the invention        from the contents of the first and second vessels;    -   4) instructions for administration, whereby to form a depot as        described herein.

Preferred Features and Combinations

In combination with the features and preferred features indicatedherein, the pre-formulations of the invention may have one or more ofthe following preferred features independently or in combination:

All proportions indicated herein may optionally be varied by up to 10%of the amount specified, optionally and preferably by up to 5%;

Component a) comprises, consists essentially of or preferably consistsof GDO;

Component b) comprises, consists essentially of or preferably consistsof soy PC;

Component c) comprises, consists essentially of or preferably consistsof a 1, 2, 3 or 4 carbon alcohol, preferably isopropanol or morepreferably ethanol;

Component c) includes a polar co-solvent such as propylene glycol;

The pre-formulation contains octreotide;

The pre-formulation contains at least one somatostatin receptor agonist(as described herein) which has agonistic and/or antagonistic effect atat least one of the SST(1)-SST(5) receptors (e.g. in humans);

The pre-formulation has a low viscosity as indicated herein;

The pre-formulation forms a non-lamellar liquid crystalline phase asindicated herein upon in vivo administration;

The pre-formulation generates a depot following in vivo administration,which depot releases at least one active agent at a therapeutic levelover a period of at least 7 days, preferably at least 21 days, morepreferably at least 28 days;

In combination with the features and preferred features indicatedherein, the method(s) of treatment of the present invention may have oneor more of the following preferred features independently or incombination:

The method comprises the administration of at least one formulation withone or more preferred features as indicated above;

The method comprises the administration of at least one formulation asindicated herein by i.m., s.c. (e.g. deep s.c.) injection;

The method comprises administration by means of a pre-filledadministration device as indicated herein;

The method comprises administration through a needle no larger than 20gauge, preferably smaller than 20 gauge, and most preferably 22 gauge,23 gauge or smaller;

The method comprises a single administration every 5 to 90 days,preferably 6 to 32 days (for example 7 days or 28-31 days).

In combination with the features and preferred features indicatedherein, the use(s) of the pre-formulations indicated herein in themanufacture of medicaments may have one or more of the followingpreferred features independently or in combination:

The use comprises the use of at least one formulation with one or morepreferred features as indicated above;

The use comprises the manufacture of a medicament for administration ofat least one formulation as indicated herein by i.m. or s.c. injection;

The use comprises the manufacture of a medicament for administration bymeans of a pre-filled administration device as indicated herein;

The use comprises the manufacture of a medicament for administrationthrough a needle no larger than 20 gauge, preferably smaller than 20gauge, and most preferably 22 gauge, 23 gauge or smaller;

The use comprises the manufacture of a medicament for administrationonce every 5 to 90 days, preferably 5 to 60 days, more preferably 6 to32 days.

In combination with the features and preferred features indicatedherein, the pre-filled devices of the invention may have one or more ofthe following preferred features independently or in combination:

They contain a preferred formulation as indicated herein;

They comprise a needle smaller than 20 gauge, preferably no larger than22 gauge or no larger than 23 gauge;

They contain a peptide active agent Somatostatin receptor agonist (e.g.octreotide) at around 1 to 100 mg.

They contain a homogeneous mixture of a composition of the invention inready-to-inject form.

They contain a formulation of components a) to d) for combination withan active agent.

They contain a total volume for administration of no more than 5 ml,preferably no more than 3 ml more preferably no more than 1.5 ml.

In combination with the features and preferred features indicatedherein, the kits of the invention may have one or more of the followingpreferred features independently or in combination:

They contain a preferred formulation as indicated herein; They contain apre-filled device as indicated herein; They contain a needle smallerthan 20 gauge, preferably no larger than 22 gauge or no larger than 23gauge;

They contain a single dose of 1 to 200 mg of peptide somatostatinreceptor agonist, preferably 1 to 100 mg and more preferably 1-50 mg;

They contain octreotide, at around 1 to 100 mg;

They contain a total volume for administration of no more than 5 ml,preferably no more than 3 ml more preferably no more than 1.5 ml.

They contain instructions for administration by a route and/or at afrequency as indicated herein;

They contain instructions for administration for use in a method oftreatment as described herein.

The Invention will now be further illustrated by reference to thefollowing non-limiting Examples and the attached Figures.

EXAMPLES

Materials

All materials used in the Examples were obtained from commercial sourcesand were of pharmacopoeial grade where applicable or of the highestpurity grade available. The following abbreviations are used throughoutthe Examples:

API Active pharmaceutical ingredient

DiETA Diethanolamine

DTPA Diethylenetriaminepentaacetic (pentetic) acid

EtOH Ethanol (99.7% Ph. Eur)

EDTA Ethylenediaminetetraacetic (edetic) acid (USP/NF)

EDTA(Na) Ethylenediaminetetraacetic acid disodium dihydrate

ETA Ethanolamine (USP/NF)

FeCl₃×6H₂O Iron(III) chloride hexahydrate

GDO Glycerol dioleate (Cithrol GDO HP-SO-(LK) from Croda)

OCT(Cl) Octreotide hydrochloride

PG Propylene glycol (Ph. Eur)

SOM(Ac) Somatostatin-14 acetate

SOM(Cl) Somatostatin-14 chloride

SPC Soy phosphatidylcholine (Lipoid S100 from Lipoid)

TRIS Tris(hydroxymethyl)aminomethane

General Procedures

Preparation of EDTA and EDTA(Na) Solutions in EtOH/PG

Samples were prepared by weighing the appropriate amounts of EDTA orEDTA(Na) and alkylamine into glass vials, e.g. 15R vials, followed byaddition of organic solvent or solvent mixture (e.g EtOH/PG (50/50w/w)). Vials were sealed and placed on either a roller mixer byend-over-end rotation at ambient RT or magnetic stirrer. Duringdissolution, vials were visually inspected for undissolved EDTAparticles using ambient and cross-polarized light.

Preparation of FeCl₃×6H₂O solutions

Samples were prepared by weighing the appropriate amount of FeCl₃×6H₂Ointo sterilized glass vials followed by addition of organic solvent orsolvent mixture. Vials were sealed and placed on a roller mixer byend-over-end rotation at ambient RT until FeCl₃×6H₂O was completelydissolved.

Preparation of SOM(Cl)

For the ion-exchange process, approximately 120 g of Dowex 1×2 chlorideform (50-100 mesh) resin was mixed with an equal amount of Milliporewater, added to a 200 mL glass ion-exchange column and left toequilibrate overnight. Next day, prior to ion-exchange the Dowex matrixwas slowly washed with 900 ml Millipore water and the ion-exchangeprocess was initiated. 3.743 g of SOM(Ac) was dissolved in 112.4 gMillipore water. Freshly prepared (within approx. 30 min) SOM(Ac)solution was loaded onto the top of the ion-exchange column. The flow(at approx. 15 s/mL) was initiated and eluate fractions of 50-250 mLeach were collected by continuously rinsing the column with Milliporewater. The eluate fractions with conductivity greater than 50 μS/cm werepooled, transferred into three 1000 mL round-bottom flasks, shell-frozenin EtOH/dry-ice using Rotavapor R-200, placed to cool at −80° C. forabout 1 h and lyophilized overnight for about 36 h. The obtained amountand yield of SOM(Cl) were 3.096 g and 82.7%, respectively. The completeexchange of acetate to chloride was confirmed by determination of thetwo anions by indirect HPLC-UV.

Preparation of Lipid Formulations

Lipid placebo formulations were prepared by weighing appropriate amountsof SPC, GDO, EDTA/alkylamine solution, and FeCl₃×6H₂O (when needed)solution into sterilized glass vials. The sealed vials were then placedon a roller mixer at room temperature until mixed completely into clearhomogeneous liquid solution (<24 hours).

API-containing formulations were prepared by adding appropriate amountsof API powder to the lipid placebo formulations in sterilized glassvials. The vials were sealed and placed on a roller mixer at roomtemperature until mixed completely into clear homogeneous liquidsolution (ca. 24 hours).

As an example, EDTA and ETA (at EDTA:ETA molar ratio 1:4) were dissolvedin EtOH/PG (50/50 w/w) mixture. Then, appropriate amounts of SPC, GDO(at SPC/GDO weight ratio 50/50) and EtOH/PG/EDTA/ETA mixture wereweighed into a sterilized 20R glass vial. The sealed vial was thenplaced on a roller mixer at room temperature until mixed completely intoclear homogeneous liquid solution (<24 hours). OCT(Cl) powder was thenadded to the lipid formulations in sterilized 15R glass vial at 2.34 wt% concentration. The vial was sealed and placed on a roller mixer atroom temperature until mixed completely into clear homogeneous liquidsolution (24 hours).

Evaluation of Octreotide Stability in Lipid Formulations (TypicalMethod)

Prepared lipid peptide (e.g. octreotide) formulations as above weredivided into sterilized 2R glass vials (0.5 g of formulation per vial).The head space of the vials was ambient air, i.e., no inert atmospheresuch as nitrogen was introduced in the head space. Vials were sealed andplaced in controlled environment storage cabinets at 25° C./60% RH and40° C./75% RH. At predefined sampling points (up to three months ofstorage) two vials of each formulation and storage cabinet werewithdrawn, equilibrated to room temperature for 1 hour and analyzed forpeptide content (assay) using gradient HPLC with UV detection.

It should be noted that the filling procedure and storage conditionsensured forced degradation conditions as the head space was composed ofair rather than inert atmosphere such as nitrogen.

HPLC-UV Determination of Peptides in Lipid Formulations

Determination of peptide (e.g. octreotide, such as octreotide chloride)in lipid formulations was carried out by gradient HPLC with UVdetection. The HILIC analytical column used was a HALO Penta-HILIC 2.7μm, 150×3.0 mm. Quantification was carried out by interpolating thepeptide (e.g. octreotide) peak area obtained in lipid formulationsamples (prepared by dissolving the lipid formulation in a samplesolvent at the required target peptide concentration) into thecalibration curves generated from standard solutions containing knownconcentrations of the corresponding peptide.

A typical mobile phases used (for example with octreotide) consisted ofwater: 2M sodium chloride:acetonitrile:trifluoroacetic acid 384:16:400:1(v/v) (mobile phase A) and water:methanol:acetonitrile:trifluoroaceticacid 20:30:950:1 (v/v) (mobile phase B). The detection was carried outat 220 nm. The sample solvent used was acetonitrile:methanol (1:1, v/v);octreotide eluted after approximately 25.2 min.

Data Presentation

In the example section, in addition to absolute API assay values,results are also in some cases expressed as a Stability Index for APIassay. The Stability Index is calculated as the API assay value in theparticular formulation divided by the API assay value in the referenceformulation. Expressed in this way, Stability Index values greater than1 means improved API stability when compared to the referenceformulation.

Measurement of Vial Headspace Oxygen Concentration

Oxygen concentration in the vial headspace was measured using aPC-controlled PreSens Microx TX3 micro fiber optic oxygen transmitterequipped with a needle-type optical oxygen microsensor (NTH, 140 μm flatbroken tip). Measurements were performed by penetrating the oxygenmicrosensor through the vial rubber stopper into the vial headspace andmeasuring the oxygen concentration until a stable readout was obtained(about 1 min).

Example 1. EDTA Solubility in the Presence and Absence of Alkylamine

0.08 wt % EDTA and EDTA(Na) solutions in EtOH/PG (50/50 w/w) wereprepared in the presence and absence of ETA (Table 1). Dissolution ofEDTA and EDTA(Na) during end-over-end rotation at ambient RT wasassessed by visual inspection (ambient and crossed-polarized light) over27 days. The results show that neither the disodium salt (EDTA(Na)) northe acid form of EDTA is soluble in EtOH/PG without using ETA even after27 days of mixing. The obtained results also show that EDTA(Na) is notsoluble in EtOH/PG even in the presence of ETA whereas the acid form ofEDTA is solubilized in EtOH/PG in the presence of 4 mol ETA per 1 mol ofEDTA already after 24 hours mixing.

TABLE 1 Solubility of 0.08 wt % EDTA and EDTA(Na) in EtOH/PG in thepresence and absence of ETA ETA/EDTA Observations after mixing forSample No EDTA type (mol/mol) 24 h 27 days Sample 1 Disodium 0.00 Notsoluble Not soluble dihydrate Sample 2 Acid form 0.00 Not soluble Notsoluble Sample 3 Disodium 3.94 Not soluble Not soluble dihydrate Sample4 Acid form 3.95 Soluble Soluble

Example 2. EDTA Solubility as a Function of ETA/EDTA Molar Ratio

Table 2 summarizes results on EDTA solubility at a concentration of 0.38wt % in EtOH/PG solvent mixtures (1/1 wt/wt) as a function of ETA/EDTAmolar ratio. The only sample where EDTA was not fully dissolved was forthe lowest ETA/EDTA molar ratio. In all other samples EDTA was solubleafter 24 h end-over-end rotation mixing at ambient RT. The obtainedresults show that about 3.5 mol of ETA per 1 mol of EDTA is close to therequired minimum amount needed to solubilize EDTA in the non-aqueoussolvent used.

TABLE 2 Solubility of 0.38 wt % EDTA in EtOH/PG as a function ofETA/EDTA molar ratio. ETA/EDTA EDTA solubility Sample ID (mol/mol) (ca24 h mixing) Sample 5 2.83 Not soluble Sample 6 3.49 Soluble Sample 73.90 Soluble Sample 8 3.90 Soluble Sample 9 4.02 Soluble Sample 10 3.99Soluble Sample 11 4.30 Soluble Sample 12 4.24 Soluble Sample 13 4.45Soluble Sample 14 4.48 Soluble Sample 15 4.62 Soluble Sample 16 4.65Soluble

Example 3. EDTA Solubility as a Function of DiETA/EDTA Molar Ratio

Table 3 summarizes EDTA solubility results in EtOH/PG (1/1 wt/wt)solvent mixture at 0.38 wt % EDTA as a function of DiETA/EDTA molarratio after 24 h end-over-end rotation mixing at ambient RT. Theobtained results show that about 4.5 mol of DiETA per 1 mol of EDTA isclose to the required minimum amount needed to solubilize EDTA innon-aqueous solvent used.

TABLE 3 Solubility of 0.38 wt % EDTA in EtOH/PG as a function ofDiETA/EDTA molar ratio. DiETA/EDTA EDTA solubility Sample ID (mol/mol)(ca 24 h mixing) Sample 17 2.14 Not soluble Sample 18 2.68 Not solubleSample 19 3.20 Not soluble Sample 20 3.52 Almost fully soluble Sample 213.97 Soluble or almost fully soluble Sample 22 4.51 Soluble Sample 235.09 Soluble

Example 4. EDTA Solubility as a Function of Ethylenediamine/EDTA MolarRatio

Table 4 summarizes EDTA solubility results in EtOH/PG (1/1 wt/wt)solvent mixture at 0.38 wt % EDTA as a function of ethylenediamine/EDTAmolar ratio after 24 h end-over-end rotation mixing at ambient RT. Theobtained results showed that about 2.5 mol of ethylenediamine per 1 molof EDTA is close to the required minimum amount needed to solubilizeEDTA in non-aqueous solvent used.

TABLE 4 Solubility of 0.38 wt % EDTA in EtOH/PG as a function ofethylenediamine/EDTA molar ratio. Ethylenediamine/EDTA EDTA solubilitySample ID (mol/mol) (ca 24 h mixing) Sample 24 1.96 Not soluble Sample25 2.45 Soluble Sample 26 3.09 Soluble Sample 27 3.46 Soluble Sample 283.92 Soluble Sample 29 4.47 Soluble Sample 30 5.00 Soluble

Example 5. EDTA Solubility as a Function of Serinol/EDTA Molar Ratio

Table 5 summarizes EDTA solubility results in EtOH/PG (1/1 wt/wt)solvent mixture at 0.38 wt % EDTA as a function of serinol/EDTA molarratio after 24 h end-over-end rotation mixing at ambient RT. Theobtained results showed that about 4 mol of serinol per 1 mol of EDTA isclose to the required minimum amount needed to solubilize EDTA innon-aqueous solvent used.

TABLE 5 Solubility of 0.38 wt % EDTA in EtOH/PG as a function ofserinol/EDTA molar ratio. Serinol/EDTA EDTA solubility Sample ID(mol/mol) (ca 24 h mixing) Sample 31 1.88 Not soluble Sample 32 2.36 Notsoluble Sample 33 3.32 Not soluble Sample 34 3.48 Almost soluble Sample35 4.11 Soluble Sample 36 4.77 Soluble Sample 37 5.09 Soluble Sample 385.45 Soluble

Example 6. EDTA Solubility as a Function of TRIS/EDTA Molar Ratio

Table 6 summarizes EDTA solubility results in EtOH/PG (1/1 wt/wt)solvent mixture at 0.38 wt % EDTA as a function of TRIS/EDTA molar ratioafter 7 days end-over-end rotation mixing at ambient RT. The obtainedresults showed that about 5 mol of TRIS per 1 mol of EDTA is close tothe required minimum amount needed to solubilize EDTA in non-aqueoussolvent used.

TABLE 6 Solubility of 0.38 wt % EDTA in EtOH/PG as a function ofTRIS/EDTA molar ratio. TRIS/EDTA EDTA solubility Sample ID (mol/mol) (ca7 days mixing) Sample 39 2.03 Not soluble Sample 40 2.57 Not solubleSample 41 2.96 Not soluble Sample 42 3.52 Not soluble Sample 43 3.97Almost soluble Sample 44 4.49 Almost soluble Sample 45 5.04 SolubleSample 46 4.98 Soluble Sample 47 5.55 Soluble Sample 48 5.98 SolubleSample 49 6.48 Soluble Sample 50 6.97 Soluble Sample 51 7.47 SolubleSample 52 8.03 Soluble

Example 7. Stability of OCT(Cl) in Lipid Formulations in the Presence ofEDTA

Lipid formulations containing 2.34 wt % of OCT(Cl) in the presence andabsence of 100 ppm of EDTA were prepared according to the compositionsgiven in Table 7. Formulations were divided into sterilized 2R glassvials (0.5 g of formulation per vial), sealed and placed in controlledenvironment storage cabinets at either 40° C./75% RH or 25° C./60% RH.The headspace of the vials was ambient air to ensure forced degradationconditions, i.e., no inert atmosphere such as nitrogen was introduced.At predefined sampling points (up to three months of storage), two vialsof each formulation and storage condition were withdrawn from thecontrolled environment cabinets, equilibrated to room temperature for 1hour and analyzed for peptide content (assay) using gradient HPLC withUV detection.

TABLE 7 OCT(Cl) containing FluidCrystal ® formulation compositions (inwt %) with and without EDTA. Sample ID OCT(Cl) SPC GDO EtOH PG ETA EDTASample 53 2.34 42.33 42.33 6.50 6.50 — — Sample 54 2.34 42.32 42.32 6.506.50 0.01 0.01

Samples of the two formulations were placed on stability as describedunder General Procedures. It should be noted that the filling procedureand storage conditions ensured forced degradation conditions as the headspace was composed of air rather than inert atmosphere such as nitrogen.FIG. 1 presents the octreotide assay at different storage time pointsand storage conditions. As shown in FIG. 1, the presence of 0.01 wt %(100 ppm) of EDTA solubilized in the lipid formulation by the use of0.01 wt % (100 ppm) ETA dramatically enhanced the peptide stability atboth storage conditions.

Example 8. Effect of EDTA Concentration on Peptide Stability

Lipid formulations containing 2.27 wt % of OCT(Cl) and differentconcentrations of EDTA were prepared according to the compositions givenin Table 8. Formulations were divided into sterilized 2R glass vials(0.5 g of formulation per vial), sealed and placed in controlledenvironment storage cabinets at either 40° C./75% RH or 25° C./60% RH.The head space of the vials was ambient air to ensure forced degradationconditions, i.e., no inert atmosphere such as nitrogen was introduced.At predefined sampling points (up to six months of storage) two vials ofeach formulation and storage condition were withdrawn from thecontrolled environment cabinets, equilibrated to room temperature for 1hour and analyzed for peptide content (assay) using gradient HPLC withUV detection.

TABLE 8 Formulation compositions with different concentrations of EDTA(all components in wt %) comprising 2.27 wt % OCT(Cl). Sample ID OCT(Cl)SPC GDO EtOH PG ETA EDTA Sample 55 2.27 42.37 42.37 6.50 6.50 — — Sample56 2.27 42.36 42.36 6.50 6.50 0.004 0.005 Sample 57 2.27 42.36 42.366.50 6.50 0.008 0.010 Sample 58 2.27 42.34 42.34 6.50 6.50 0.021 0.025Sample 59 2.27 42.32 42.32 6.50 6.50 0.042 0.050 Sample 60 2.27 42.3042.30 6.50 6.50 0.063 0.075

Samples of the six formulations were placed on stability as describedunder General Procedures. It should be noted that the filling procedureand storage conditions ensured forced degradation conditions as the headspace was composed of air rather than inert atmosphere such as nitrogen.The results are shown in FIG. 2. As shown, the presence of EDTAsolubilized in the lipid formulation with the help of ETA dramaticallyenhanced the peptide stability vs. the reference formulation notcontaining EDTA/ETA. The maximum stabilization effect was achievedwithin the concentration interval 50-250 ppm (0.005-0.025 wt %) EDTA.

Example 9. Long-Term Stability of OCT(Cl) in Lipid Formulations in thePresence of EDTA

Lipid formulations containing OCT(Cl) in the absence and presence of 100ppm EDTA were prepared according to the compositions given in Table 9.Formulations were divided into sterilized 1 mL 22G×½″ glass syringes(Schott AG) (0.5 g of formulation per syringe), sealed with plunger andplaced in a controlled environment storage cabinet at 25° C./60% RH. Atpredefined sampling points (up to twelve months of storage), twosyringes of each formulation were withdrawn from the controlledenvironment cabinet, equilibrated to room temperature for 1 hour andanalyzed for peptide content (assay) using gradient HPLC with UVdetection.

TABLE 9 OCT(Cl) containing lipid formulation compositions (in wt %)without and with EDTA. The octreotide content corresponds to 20 mg/mLoctreotide free base when corrected for peptide content, purity andformulation density. Sample ID OCT(Cl) SPC GDO EtOH PG ETA EDTA Sample61 2.27 42.37 42.37 6.50 6.50 — — Sample 62 2.27 42.36 42.36 6.50 6.500.008 0.010

FIG. 3 presents the octreotide assay at different storage time points.As shown, the presence of 0.01 wt % (100 ppm) of EDTA solubilized in thelipid formulation with the help of ETA significantly enhanced thelong-term peptide stability in pre-filled syringes at the long-term 25°C./60% RH storage condition.

Example 10. Stability of OCT(Cl) in Lipid Formulations in the Presenceof Iron and EDTA

Lipid formulations containing OCT(Cl) and different amounts of Fe³⁺ andEDTA were prepared according to the compositions given in Table 10.Formulations were divided into sterilized 2R glass vials (0.5 g offormulation per vial), sealed and placed in a controlled environmentstorage cabinet at 40° C./75% RH. The head space of the vials wasambient air to ensure forced degradation conditions, i.e., no inertatmosphere such as nitrogen was introduced. At 1-month sampling pointtwo vials of each formulation and storage condition were withdrawn fromthe controlled environment cabinet, equilibrated to room temperature for1 hour and analyzed for peptide content (assay) using gradient HPLC withUV detection.

TABLE 10 OCT(Cl) containing FluidCrystal ® formulation compositions (inwt %) with different concentrations of Fe³⁺ and EDTA. The octreotidecontent corresponds to 20 mg/mL octreotide free base when corrected forpeptide content, purity and formulation density. The SPC/GDO weightratio is 50/50 in all formulations. Sample ID OCT(Cl) SPC + GDO EtOH PGEDTA ETA FeCl₃ × 6H₂O* Sample 63 2.27 84.73000 6.50 6.50 — — — Sample 642.27 84.72903 6.50 6.50 — — 0.00097 Sample 65 2.27 84.72758 6.50 6.50 —— 0.00242 Sample 66 2.27 84.72516 6.50 6.50 — — 0.00484 Sample 67 2.2784.72541 6.50 6.50 0.00250 0.00209 — Sample 68 2.27 84.72444 6.50 6.500.00250 0.00209 0.00097 Sample 69 2.27 84.72299 6.50 6.50 0.002500.00209 0.00242 Sample 70 2.27 84.72057 6.50 6.50 0.00250 0.002090.00484 Sample 71 2.27 84.71164 6.50 6.50 0.01000 0.00836 — Sample 722.27 84.71067 6.50 6.50 0.01000 0.00836 0.00097 Sample 73 2.27 84.709226.50 6.50 0.01000 0.00836 0.00242 Sample 74 2.27 84.70680 6.50 6.500.01000 0.00836 0.00484 Sample 75 2.27 84.68409 6.50 6.50 0.025000.02091 — Sample 76 2.27 84.68312 6.50 6.50 0.02500 0.02091 0.00097Sample 77 2.27 84.68167 6.50 6.50 0.02500 0.02091 0.00242 Sample 78 2.2784.67925 6.50 6.50 0.02500 0.02091 0.00484 *0.00097, 0.00242 and 0.00484wt % of FeCl₃ × 6H₂O corresponds to 2, 5 and 10 ppm of Fe³⁺,respectively.

FIG. 4 presents the octreotide assay at 1-month time point as a functionof Fe³⁺ concentration in the presence of different amounts of EDTA. Asevident, with increasing the Fe³⁺ concentration, more EDTA is needed toprotect OCT from degradation. The protection against OCT degradation inthe presence of Fe³⁺ is enhanced with increasing EDTA concentration upto 100 ppm, followed by some decline between 100 and 250 ppm. There isalso a clear correlation between Fe³⁺ concentration and amount of EDTAneeded to suppress the catalytic activity of iron. As shown in FIG. 5, amaximum stabilization effect is achieved starting from EDTA:Fe³⁺ molarratio of about 2:1. This corresponds to about 100 ppm EDTA at a Fe³⁺content of 10 ppm.

Example 11. Stability of OCT(Cl) in Lipid Formulations with EDTA andIron in the Absence and Presence of ETA

Lipid formulations containing EDTA or EDTA(Na) in the absence andpresence of ETA were prepared according to the compositions given inTable 11. As shown in Example 1, neither EDTA(Na) nor EDTA are solublein EtOH/PG without using ETA. EDTA(Na) was also insoluble in EtOH/PGeven in the presence of ETA as assessed by visual inspection. Therefore,EDTA(Na), EDTA and EDTA(Na)/ETA containing mixtures in EtOH/PG wereadditionally filtered using a Millex-LG hydrophilic PTFE 0.2 μm syringefilter to remove the non-dissolved EDTA particles. After preparation,formulations were divided into sterilized 2R glass vials (0.5 g offormulation per vial), sealed and placed in a controlled environmentstorage cabinet at 40° C./75% RH. The headspace of the vials was ambientair to ensure forced degradation conditions, i.e., no inert atmospheresuch as nitrogen was introduced. At predefined sampling points (up totwo months of storage) two vials of each formulation and storagecondition were withdrawn from controlled environment cabinets,equilibrated to room temperature for 1 hour and analyzed for peptidecontent (assay) using gradient HPLC with UV detection.

TABLE 11 OCT(Cl) containing lipid formulation compositions (in wt %)with different concentrations of Fe³⁺ and EDTA. The octreotide contentcorresponds to 20 mg/mL octreotide free base when corrected for peptidecontent, purity and formulation density. The SPC/GDO weight ratio was50/50 in all formulations. Sample ID OCT (Cl) SPC + GDO EtOH PG EDTAEDTA(Na) ETA FeCl₃ × 6H₂O ** Sample 79 2.27 84.73 6.50 6.50 — — — —Sample 80* 2.27 84.72 6.50 6.50 — 0.01 — 0.00242 Sample 81* 2.27 84.716.50 6.50 — 0.01 0.00840 0.00242 Sample 82* 2.27 84.72 6.50 6.50 0.01 —— 0.00242 Sample 83 2.27 84.71 6.50 6.50 0.01 — 0.00840 0.00242 *Forpreparation of these formulations, EDTA mixtures in EtOH/PG werefiltered using Millex-LG hydrophilic PTFE 0.2 μm syringe filter toremove insoluble EDTA particles. ** 0.00242 wt % of FeCl₃ × 6H₂Ocorresponds to 5 ppm of Fe³⁺.

FIG. 6 presents the assay and Stability Index values of octreotide as afunction of time, respectively. As seen, only EDTA solubilized in thelipid formulation with the help of ETA dramatically enhanced the peptidestability compared to the reference formulation in the presence of 5 ppmFe³⁺. Under the same conditions, formulations containing EDTA(Na), EDTAor EDTA(Na)/ETA showed negative effect on OCT(Cl) stability (vs. thereference formulation).

Example 12. Effect of Different Alkylamines and Solvents on Stability ofOCT(Cl) in Lipid Formulations with EDTA

Lipid formulations were prepared according to the compositions given inTable 12. Formulations were divided into sterilized 2R glass vials (0.5g of formulation per vial), sealed and placed in a controlledenvironment storage cabinet at 40° C./75% RH. The headspace of the vialswas ambient air to ensure forced degradation conditions, i.e., no inertatmosphere such as nitrogen was introduced. At predefined samplingpoints (up to two months of storage) two vials of each formulation andstorage condition were withdrawn from the controlled environmentcabinets, equilibrated to room temperature for 1 hour and analyzed forpeptide content (assay) using gradient HPLC with UV detection.

TABLE 12 OCT(Cl) containing lipid formulation compositions (in wt %).The octreotide content corresponds to 20 mg/mL octreotide free base whencorrected for peptide content, purity and formulation density. TheSPC/GDO weight ratio was 50/50 and ETA:EDTA, DiETA:EDTA andethylenediamine:EDTA molar ratios were 4:1 in all formulations. EthyleneSample ID OCT (Cl) SPC + GDO EtOH PG EDTA ETA DiETA diamine Sample 842.27 84.71160 6.50 6.50 0.01 0.0084 — — Sample 85 2.27 84.70560 6.506.50 0.01 — 0.01440 — Sample 86 2.27 84.71180 6.50 6.50 0.01 — — 0.00820

FIG. 7 presents the octreotide assay at different storage time points.As shown, the different alkylamines (ETA, DiETA or ethylenediamine) usedto solubilize 0.01 wt % (100 ppm) of EDTA into the lipid formulationsenhanced the peptide stability to a similar high degree when compared tothe reference formulation. The obtained results also show that thepositive effect of EDTA on the stability of OCT(Cl) is independent onthe mixture used to prepare the lipid formulations as indicated by thedata for EtOH/PG containing formulations in FIG. 8 when compared withFIG. 11.

Example 13. Stability of SOM(Cl) in Lipid Formulations in the Presenceof EDTA

Lipid formulations containing SOM(Cl) in the absence and presence of 100ppm EDTA using EtOH/PG were prepared according to the compositions givenin Table 13. Formulations were divided into sterilized 2R glass vials(0.5 g of formulation per vial), sealed and placed in controlledenvironment storage cabinets at either 40° C./75% RH or 25° C./60% RH.The head space of the vials was ambient air to ensure forced degradationconditions, i.e., no inert atmosphere such as nitrogen was introduced.At predefined sampling points (up to three months of storage) two vialsof each formulation and storage condition were withdrawn from thecontrolled environment cabinets, equilibrated to room temperature for 1hour and analyzed for peptide content (assay) using gradient HPLC withUV detection.

TABLE 13 SOM(Cl) containing lipid formulation compositions (in wt %)without and with EDTA. The SPC/GDO weight ratio was 50/50 in allformulations. Sample ID SOM(Cl) SPC + GDO EtOH PG EDTA ETA Sample 892.00 86.00000 10.00 2.00 — — Sample 90 2.00 85.98164 10.00 2.00 0.010000.00836

FIG. 9 presents the SOM assay at different storage time points andstorage conditions. As shown, independently of solvent mixture used toprepare the lipid formulations, the presence of 100 ppm of EDTAsolubilized in the lipid formulation by the use of ETA dramaticallyenhanced the peptide stability at both 40° C./75% RH and 25° C./60% RHstorage conditions.

Example 14. Lipid Oxidation in Placebo Lipid Formulations in thePresence of EDTA

Lipid placebo formulations in the absence and presence of 100 ppm EDTAwere prepared according to the compositions given in Table 14.Formulations were divided into sterilized 2R glass vials (1 g offormulation per vial), sealed and placed in controlled environmentstorage cabinets at either 60° C./ambient RH or 40° C./75% RH. Theheadspace of the vials was ambient air to ensure forced lipid oxidationconditions, i.e., no inert atmosphere such as nitrogen was introduced.Some formulations also contained 5 ppm Fe³⁺ to enhance the oxidativestress conditions (Table 14). At predefined sampling points (up to 9days of storage at 60° C./ambient RH and up to 30 days of storage at 40°C./75% RH) two vials of each formulation were withdrawn from thecontrolled environment cabinets, equilibrated to room temperature for 1hour and analyzed for oxygen concentration in the vial headspace (oxygenconsumption is here used as an indirect measure of lipid oxidation inthe lipid formulations) using a needle-type oxygen microsensor.

TABLE 14 Lipid formulation compositions (in wt %) without and with EDTA.The SPC/GDO weight ratio was 50/50 and 35/65 in Samples 103-106 andSamples 107-110, respectively. Sample ID SPC GDO EtOH EDTA ETA* FeCl₃ ×6H₂O** Sample 103 45.00 45.00 10 — — — Sample 104 45.00 45.00 10 — —0.00242 Sample 105 44.99 44.99 10 0.01 0.0116 — Sample 106 44.99 44.9910 0.01 0.0116 0.00242 Sample 107 31.50 58.50 10 — — — Sample 108 31.5058.50 10 — — 0.00242 Sample 109 31.49 58.49 10 0.01 0.0116 — Sample 11031.49 58.49 10 0.01 0.0116 0.00242 *ETA:EDTA molar ratio is 5.5:1**0.00242 wt % of FeCl₃ × 6H₂O corresponds to 5 ppm of Fe³⁺

Example 15. DTPA Solubility as a Function of ETA/DTPA Molar Ratio

0.08 wt % DTPA solutions in EtOH/PG (50/50 w/w) were prepared in theabsence and presence of various amounts of ETA added at differentETA/DTPA molar ratios (Table 15). The results show that DTPA is notsoluble in EtOH/PG without using ETA. The obtained results also showthat about 4.3 mol of ETA per 1 mol of DTPA is close to the requiredminimum amount needed to solubilize DTPA in the non-aqueous solventused.

TABLE 15 Solubility of 0.08 wt % DTPA in EtOH/PG as a function ofETA/DTPA molar ratio. ETA/DTPA DTPA solubility Sample ID (mol/mol) (ca24 h mixing) Sample 111 0.0 Not soluble Sample 112 1.7 Not solubleSample 113 4.3 Soluble Sample 114 4.8 Soluble Sample 115 6.2 SolubleSample 116 7.9 Soluble

1. A pre-formulation comprising: a) at least one diacyl glycerol; b) at least one phospholipid selected from the group consisting of a phosphatidyl choline (PC), a phosphatidyl ethanolamine (PE), and a phosphatidyl inositol (PI); c) at least one biocompatible, organic solvent selected from the group consisting of ethanol, propanol, isopropanol, benzyl alcohol, a polar co-solvent, and mixtures thereof; d) an alkyl ammonium EDTA salt comprising an anion of ethylenediaminetetraacetic acid or an analogue thereof; and e) at least one somatostatin receptor agonist selected from octreotide or a salt thereof; wherein the pre-formulation has a water content in the range of 0 to 1.0 wt %.
 2. A pre-formulation of claim 1 wherein component a) comprises glycerol dioleate (GDO).
 3. (canceled)
 4. A pre-formulation of claim 1 wherein component b) comprises a phosphatidyl choline (PC).
 5. (canceled)
 6. A pre-formulation of claim 1 wherein the alkylammonium EDTA is a salt of ethanolamine (ETA) and EDTA.
 7. A pre-formulation of claim 6 wherein the equivalents of ETA relative to the amount of EDTA is in the range of 3.5 to 7 (mol/mol).
 8. A pre-formulation of claim 1 wherein the alkyl ammonium EDTA salt is present in an amount of 0.001 to 0.050 wt % of the pre-formulation (10-500 ppm) based on the amount of EDTA free acid.
 9. (canceled)
 10. A pre-formulation of claim 1 wherein the somatostatin receptor agonist comprises octreotide chloride.
 11. A pre-formulation of claim 1 wherein component a) is present at a level of 20-60% by weight.
 12. A pre-formulation of claim 1 wherein component b) is present at a level of 20-60% by weight.
 13. (canceled)
 14. A pre-formulation of claim 1 wherein component c) comprises a mixture of ethanol and propylene glycol.
 15. A pre-formulation of claim 1 wherein component c) is present at a level of 2 to 20% by weight.
 16. A pre-formulation of claim 1 wherein component c) includes 2 to 12 wt. % of propylene glycol.
 17. (canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. A composition formed by exposure of a pre-formulation according to claim 1 with excess aqueous fluid having a liquid crystalline phase structure.
 22. (canceled)
 23. A medicament comprising the pre-formulation of claim
 1. 24. A method for the treatment of a human or non-human mammalian subject comprising administering to the subject a pre-formulation of claim
 1. 25. The method of claim 24, wherein the pre-formulation is administered to the human or non-human mammalian subject in need thereof to treat at least one condition selected from the group consisting of acromegaly, cancers, carcinomas, melanomas, tumours expressing at least one somatostatin receptor, sst(2)-positive tumours, sst(5)-positive tumours, prostate cancers, gastro-entero-pancreatic endocrine tumours, gastro-entero-pancreatic neuroendocrine (GEP NE) tumours, carcinoid tumours, insulinomas, gastrinomas, vasoactive intestinal peptide (VIP) tumours and glucagonomas, TSH-secreting pituitary adenomas, elevated growth hormone (GH), elevated insulin-like growth factor I (IGF-I), varicial bleeding, chemotherapy induced gastro intestinal problems, lymphorrhea, diabetic retinopathy, thyroid eye disease, obesity, pancreatitis, and related conditions.
 26. (canceled)
 27. (canceled)
 28. (canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled)
 32. A pre-filled administration device containing a pre-formulation of claim
 1. 33. A kit comprising an administration device of claim
 32. 34. (canceled)
 35. (canceled)
 36. A method of reducing oxidation of at least one active agent in a pre-formulation comprising: a) at least one diacyl glycerol; b) at least one phospholipid selected from the group consisting of a phosphatidyl choline (PC), a phosphatidyl ethanolamine (PE), and a phosphatidyl inositol (PI); c) at least one biocompatible, organic solvent selected from the group consisting of ethanol, propanol, isopropanol, benzyl alcohol, a polar co-solvent, and mixtures thereof; and e) at least one somatostatin receptor agonist selected from octreotide or a salt thereof; wherein the pre-formulation has a water content in the range of 0 to 1.0 wt %; the method comprising including an alkylammonium EDTA salt comprising an anion of ethylenediaminetetraacetic acid or an analogue thereof in the pre-formulation.
 37. (canceled)
 38. A process for preparing a pre-formulation of claim 1 comprising the steps of dispersing EDTA or a hydrate thereof and an alkylamine in a biocompatible organic solvent to produce a dispersion; mixing the dispersion until the EDTA and alkylamine are fully dissolved to produce a mixture; and adding the at least one diacyl glycerol, the at least one phospholipid, and the at least one somatostatin receptor agonist to the mixture to produce the pre-formulation.
 39. A pre-formulation of claim 1 wherein component e) is octreotide and is present at a level of 0.5-9% by weight.
 40. A pre-formulation of claim 1 wherein component e) is octreotide and is present at a level of 10 to 50 mg/mL.
 41. A pre-formulation of claim 1 wherein component e) is octreotide and is present at a level of 10 to 30 mg/mL.
 42. A pre-formulation comprising: a) glycerol dioleate (GDO); b) phosphatidyl choline (PC); c) ethanol and propylene glycol (PG); d) an alkyl ammonium EDTA salt; and e) octreotide or a salt thereof.
 43. The pre-formulation of claim 42 wherein the pre-formulation comprises: a) 33-43% by weight of GDO; b) 33-55% by weight of PC; c) 8-18% by weight of a mixture of ethanol and PG; d) 10-200 ppm of an alkyl ammonium EDTA salt; and e) 0.1-12% by weight of octreotide or a salt thereof.
 44. The pre-formulation of claim 43 wherein component d) is a salt of ethanolamine (ETA) and EDTA.
 45. The pre-formulation of claim 43 wherein component e) is octreotide chloride. 